Calcium binding protein

ABSTRACT

The invention provides a human calcium binding protein (hCBP) and polynucleotides which identify and encode hCBP. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating, or preventing disorders associated with expression of hCBP.

[0001] This application is a divisional application of U.S. applicationSer. No. 09/470,253, filed Dec. 22, 1999, which is a divisionalapplication of U.S. application Ser. No. 09/190,965, filed Nov. 13,1998, now U.S. Pat. No. 6,071,721, issued Jun. 6, 2000, both entitledCALCIUM BINDING PROTEIN, all of which applications and patents arehereby incorporated herein by reference.

FIELD OF THE INVENTION

[0002] This invention relates to nucleic acid and amino acid sequencesof a calcium binding protein and to the use of these sequences in thediagnosis, treatment, and prevention of cancer, reproductive disorders,immune disorders, and developmental disorders.

BACKGROUND OF THE INVENTION

[0003] In nearly all eukaryotic cells, calcium (Ca²⁺) functions as anintracellular signaling molecule in diverse cellular processes includingcell proliferation, neurotransmitter secretion, glycogen metabolism, andskeletal muscle contraction. Within a resting cell, the concentration ofCa²⁺ in the cytosol is extremely low, <10⁻⁷ M. However, when the cell isstimulated by an external signal, such as a neural impulse or a growthfactor, the cytosolic concentration of Ca²⁺ increases by about 50-fold.This influx of Ca²⁺ is caused by the opening of plasma membrane Ca²⁺channels and the release of Ca²⁺ from intracellular stores such as theendoplasmic reticulum. Ca²⁺ directly activates regulatory enzymes, suchas protein kinase C, which trigger signal transduction pathways. Ca²⁺also binds to specific Ca²⁺-binding proteins (CBPs) such as calmodulin(CaM) which then activate multiple target proteins including enzymes,membrane transport pumps, and ion channels. CaM is the most widelydistributed and the most common mediator of calcium effects and appearsto be the primary sensor of Ca²⁺ changes in eukaryotic cells. Thebinding of Ca²⁺ to CaM induces marked conformational changes in theprotein permitting interaction with, and regulation of over 100different proteins. CaM interactions are involved in a multitude ofcellular processes including, but not limited to, gene regulation, DNAsynthesis, cell cycle progression, mitosis, cytokinesis, cytoskeletalorganization, muscle contraction, signal transduction, ion homeostasis,exocytosis, and metabolic regulation (Celio, M. R. et al. (1996)Guidebook to Calcium-binding Proteins, Oxford University Press, Oxford,UK, pp. 15-20).

[0004] A novel mouse gene named MO25, expressed during early stages ofdevelopment, has recently been identified and is believed to encode aCBP. The Drosophila equivalent of MO25, DMO25, encodes a polypeptide of339 amino acid residues with a calculated molecular mass of 39.3 kDa.The novel CBP was found to be conserved among Drosophila, mouse, andyeast. In particular, the carboxy-terminal region of the protein ishighly conserved among these species. A homology search revealed thatthe amino acid sequence of MO25 and DMO25 is similar to a proteinencoded in an open reading frame near the calcineurin B subunit gene onchromosome XI in Saccharomyces cerevisiae. Calcineurin B is the smallCa²⁺-binding regulatory subunit of calcineurin, a CaM-regulated proteinphosphatase. The conservation of the MO25 and DMO25 gene structure amongspecies and the wide tissue expression profile indicates that thefunction of the gene is likely to be fundamental in many cell types aswell as during development (Nozaki, M. et al. (1996) DNA Cell Biol.15:505-509; and Miyamoto, H. et al. (1993) Mol. Reprod. Dev. 34:1-7).

[0005] CBPs are implicated in a variety of disorders. For example,calcineurin is found in the cells of all eukaryotes ranging from yeastto mammals. Calcineurin is a target for inhibition by theimmunosuppressive agents cyclosporin A and FK506 emphasizing itsimportance in immune disorders (Kissinger, C. R. et al. (1995) Nature378:641-644). Calcineurin also plays a critical role in transcriptionalregulation and growth control in T-lymphocytes (Wang, M. G. et al.(1996) Cytogenet. Cell Genet. 72:236-241). Additionally, levels of CaMare increased several-fold in tumors and tumor-derived cell lines forvarious types of cancer (Rasmussen, C. D. and Means, A. R. (1989) Trendsin Neuroscience 12:433-438). Calcium binding S100β is another example ofa CBP involved in a variety of disorders. S100β contains an EF-handmotif and is abundantly expressed in the nervous system. S100β levelsare increased in the blood and cerebrospinal fluid of patients withneurological injury resulting from cerebral infarction, transientischemic attacks, hemorrhagia, head trauma, and Down's syndrome.Furthermore, S100β and other neural-specific CBPs may also protectagainst neurodegenerative disorders, such as Alzheimer's, Parkinson's,and Huntington's diseases.

[0006] The discovery of a new calcium binding protein and thepolynucleotides encoding it satisfies a need in the art by providing newcompositions which are useful in the diagnosis, prevention, andtreatment of cancer, reproductive disorders, immune disorders, anddevelopmental disorders.

SUMMARY OF THE INVENTION

[0007] The invention is based on the discovery of a new human calciumbinding protein (hCBP), the polynucleotides encoding hCBP, and the useof these compositions for the diagnosis, treatment, or prevention ofcancer, reproductive disorders, immune disorders, and developmentaldisorders.

[0008] The invention features a substantially purified polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQID NO:1.

[0009] The invention further provides a substantially purified varianthaving at least 90% amino acid sequence identity to the amino acidsequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention alsoprovides an isolated and purified polynucleotide encoding thepolypeptide comprising the amino acid sequence of SEQ ID NO:1 or afragment of SEQ ID NO:1. The invention also includes an isolated andpurified polynucleotide variant having at least 70% polynucleotidesequence identity to the polynucleotide encoding the polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQID NO:1.

[0010] The invention further provides an isolated and purifiedpolynucleotide which hybridizes under stringent conditions to thepolynucleotide encoding the polypeptide comprising the amino acidsequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as anisolated and purified polynucleotide having a sequence which iscomplementary to the polynucleotide encoding the polypeptide comprisingthe amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.

[0011] The invention also provides an isolated and purifiedpolynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 ora fragment of SEQ ID NO:2, and an isolated and purified polynucleotidevariant having at least 70% polynucleotide sequence identity to thepolynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 ora fragment of SEQ ID NO:2. The invention also provides an isolated andpurified polynucleotide having a sequence complementary to thepolynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 ora fragment of SEQ ID NO:2.

[0012] The invention also provides a method for detecting apolynucleotide in a sample containing nucleic acids, the methodcomprising the steps of (a) hybridizing the complement of thepolynucleotide sequence to at least one of the polynucleotides of thesample, thereby forming a hybridization complex; and (b) detecting thehybridization complex, wherein the presence of the hybridization complexcorrelates with the presence of a polynucleotide in the sample. In oneaspect, the method further comprises amplifying the polynucleotide priorto hybridization.

[0013] The invention further provides an expression vector containing atleast a fragment of the polynucleotide encoding the polypeptidecomprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. Inanother aspect, the expression vector is contained within a host cell.

[0014] The invention also provides a method for producing a polypeptide,the method comprising the steps of: (a) culturing the host cellcontaining an expression vector containing at least a fragment of apolynucleotide under conditions suitable for the expression of thepolypeptide; and (b) recovering the polypeptide from the host cellculture.

[0015] The invention also provides a pharmaceutical compositioncomprising a substantially purified polypeptide having the sequence ofSEQ ID NO:1 or a fragment of SEQ ID NO:1 in conjunction with a suitablepharmaceutical carrier.

[0016] The invention further includes a purified antibody which binds toa polypeptide comprising the sequence of SEQ ID NO:1 or a fragment ofSEQ ID NO:1, as well as a purified agonist and a purified antagonist ofthe polypeptide.

[0017] The invention also provides a method for treating or preventing adisorder associated with decreased expression or activity of hCBP, themethod comprising administering to a subject in need of such treatmentan effective amount of a pharmaceutical composition comprising asubstantially purified polypeptide having the amino acid sequence of SEQID NO:1 or a fragment of SEQ ID NO:1, in conjunction with a suitablepharmaceutical carrier.

[0018] The invention also provides a method for treating or preventing adisorder associated with increased expression or activity of hCBP, themethod comprising administering to a subject in need of such treatmentan effective amount of an antagonist of the polypeptide having the aminoacid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.

BRIEF DESCRIPTION OF THE FIGURES AND TABLE

[0019]FIGS. 1A, 1B, 1C and 1D show the amino acid sequence (SEQ ID NO:1)and nucleic acid sequence (SEQ ID NO:2) of hCBP. The alignment wasproduced using MACDNASIS PRO software (Hitachi Software Engineering,South San Francisco, Calif.).

[0020]FIGS. 2A and 2B show the amino acid sequence alignment betweenhCBP (3734805; SEQ ID NO:1), MO25 (GI 262934; SEQ ID NO:3), DMO25 (GI1794137; SEQ ID NO:4), and yeast-like CBP (GI 1255838; SEQ ID NO:5)produced using the multisequence alignment program of LASERGENE software(DNASTAR, Madison Wis.).

[0021] Table 1 shows the programs, their descriptions, references, andthreshold parameters used to is analyze hCBP.

DESCRIPTION OF THE INVENTION

[0022] Before the present proteins, nucleotide sequences, and methodsare described, it is understood that this invention is not limited tothe particular machines, materials and methods described, as these mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims.

[0023] It must be noted that as used herein and in the appended claims,the singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, a referenceto “a host cell” includes a plurality of such host cells, and areference to “an antibody” is a reference to one or more antibodies andequivalents thereof known to those skilled in the art, and so forth.

[0024] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any machines,materials, and methods similar or equivalent to those described hereincan be used to practice or test the present invention, the preferredmachines, materials and methods are now described. All publicationsmentioned herein are cited for the purpose of describing and disclosingthe cell lines, protocols, reagents and vectors which are reported inthe publications and which might be used in connection with theinvention. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

[0025] Definitions

[0026] “hCBP” refers to the amino acid sequences of substantiallypurified hCBP obtained from any species, particularly a mammalianspecies, including bovine, ovine, porcine, murine, equine, andpreferably the human species, from any source, whether natural,synthetic, semi-synthetic, or recombinant.

[0027] The term “agonist” refers to a molecule which, when bound tohCBP, increases or prolongs the duration of the effect of hCBP. Agonistsmay include proteins, nucleic acids, carbohydrates, or any othermolecules which bind to and modulate the effect of hCBP.

[0028] An “allelic variant” is an alternative form of the gene encodinghCBP. Allelic variants may result from at least one mutation in thenucleic acid sequence and may result in altered mRNAs or in polypeptideswhose structure or function may or may not be altered. Any given naturalor recombinant gene may have none, one, or many allelic forms. Commonmutational changes which give rise to allelic variants are generallyascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

[0029] “Altered” nucleic acid sequences encoding hCBP include thosesequences with deletions, insertions, or substitutions of differentnucleotides, resulting in a polypeptide the same as hCBP or apolypeptide with at least one functional characteristic of hCBP.Included within this definition are polymorphisms which may or may notbe readily detectable using a particular oligonucleotide probe of thepolynucleotide encoding hCBP, and improper or unexpected hybridizationto allelic variants, with a locus other than the normal chromosomallocus for the polynucleotide sequence encoding hCBP. The encoded proteinmay also be “altered,” and may contain deletions, insertions, orsubstitutions of amino acid residues which produce a silent change andresult in a functionally equivalent hCBP. Deliberate amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues, as long as the biological orimmunological activity of hCBP is retained. For example, negativelycharged amino acids may include aspartic acid and glutamic acid,positively charged amino acids may include lysine and arginine, andamino acids with uncharged polar head groups having similarhydrophilicity values may include leucine, isoleucine, and valine;glycine and alanine; asparagine and glutamine; serine and threonine; andphenylalanine and tyrosine.

[0030] The terms “amino acid” or “amino acid sequence” refer to anoligopeptide, peptide, polypeptide, or protein sequence, or a fragmentof any of these, and to naturally occurring or synthetic molecules. Inthis context, “fragments,” “immunogenic fragments,” or “antigenicfragments” refer to fragments of hCBP which are preferably at least 5 toabout 15 amino acids in length, most preferably at least 14 amino acids,and which retain some biological activity or immunological activity ofhCBP. Where “amino acid sequence” is recited to refer to an amino acidsequence of a naturally occurring protein molecule, “amino acidsequence” and like terms are not meant to limit the amino acid sequenceto the complete native amino acid sequence associated with the recitedprotein molecule.

[0031] “Amplification” relates to the production of additional copies ofa nucleic acid sequence. Amplification is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art.

[0032] The term “antagonist” refers to a molecule which, when bound tohCBP, decreases the amount or the duration of the effect of thebiological or immunological activity of hCBP. Antagonists may includeproteins, nucleic acids, carbohydrates, antibodies, or any othermolecules which decrease the effect of hCBP.

[0033] The term “antibody” refers to intact molecules as well as tofragments thereof, such as Fab, F(ab′)₂, and Fv fragments, which arecapable of binding the epitopic determinant. Antibodies that bind hCBPpolypeptides can be prepared using intact polypeptides or usingfragments containing small peptides of interest as the immunizingantigen. The polypeptide or oligopeptide used to immunize an animal(e.g., a mouse, a rat, or a rabbit) can be derived from the translationof RNA, or synthesized chemically, and can be conjugated to a carrierprotein if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin, thyroglobulin, and keyholelimpet hemocyanin (KLH). The coupled peptide is then used to immunizethe animal.

[0034] The term “antigenic determinant” refers to that fragment of amolecule (i.e., an epitope) that makes contact with a particularantibody. When a protein or a fragment of a protein is used to immunizea host animal, numerous regions of the protein may induce the productionof antibodies which bind specifically to antigenic determinants (givenregions or three-dimensional structures on the protein). An antigenicdeterminant may compete with the intact antigen (i.e., the immunogenused to elicit the immune response) for binding to an antibody.

[0035] The term “antisense” refers to any composition containing anucleic acid sequence which is complementary to the “sense” strand of aspecific nucleic acid sequence. Antisense molecules may be produced byany method including synthesis or transcription. Once introduced into acell, the complementary nucleotides combine with natural sequencesproduced by the cell to form duplexes and to block either transcriptionor translation. The designation “negative” can refer to the antisensestrand, and the designation “positive” can refer to the sense strand.

[0036] The term “biologically active,” refers to a protein havingstructural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, “immunologically active” refers to thecapability of the natural, recombinant, or synthetic hCBP, or of anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

[0037] The terms “complementary” or “complementarity” refer to thenatural binding of polynucleotides by base pairing. For example, thesequence “5′ A-G-T 3′” bonds to the complementary sequence “3′ T-C-A5′.” Complementarity between two single-stranded molecules may be“partial,” such that only some of the nucleic acids bind, or it may be“complete,” such that total complementarity exists between the singlestranded molecules. The degree of complementarity between nucleic acidstrands has significant effects on the efficiency and strength of thehybridization between the nucleic acid strands. This is of particularimportance in amplification reactions, which depend upon binding betweennucleic acids strands, and in the design and use of peptide nucleic acid(PNA) molecules.

[0038] A “composition comprising a given polynucleotide sequence” or a“composition comprising a given amino acid sequence” refer broadly toany composition containing the given polynucleotide or amino acidsequence. The composition may comprise a dry formulation or an aqueoussolution. Compositions comprising polynucleotide sequences encoding hCBPor fragments of hCBP may be employed as hybridization probes. The probesmay be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., sodium dodecyl sulfate; SDS), and other components(e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).

[0039] “Consensus sequence” refers to a nucleic acid sequence which hasbeen resequenced to resolve uncalled bases, extended using XL-PCR kit(Perkin-Elmer, Norwalk Conn.) in the 5′ and/or the 3′ direction, andresequenced, or which has been assembled from the overlapping sequencesof more than one Incyte Clone using a computer program for fragmentassembly, such as the GELVIEW fragment assembly system (GCG, MadisonWis.). Some sequences have been both extended and assembled to producethe consensus sequence.

[0040] The term “correlates with expression of a polynucleotide”indicates that the detection of the presence of nucleic acids, the sameor related to a nucleic acid sequence encoding hCBP, by northernanalysis is indicative of the presence of nucleic acids encoding hCBP ina sample, and thereby correlates with expression of the transcript fromthe polynucleotide encoding hCBP.

[0041] A “deletion” refers to a change in the amino acid or nucleotidesequence that results in the absence of one or more amino acid residuesor nucleotides.

[0042] The term “derivative” refers to the chemical modification of apolypeptide sequence, or a polynucleotide sequence. Chemicalmodifications of a polynucleotide sequence can include, for example,replacement of hydrogen by an alkyl, acyl, or amino group. A derivativepolynucleotide encodes a polypeptide which retains at least onebiological or immunological function of the natural molecule. Aderivative polypeptide is one modified by glycosylation, pegylation, orany similar process that retains at least one biological orimmunological function of the polypeptide from which it was derived.

[0043] The term “similarity” refers to a degree of complementarity.There may be partial similarity or complete similarity. The word“identity” may substitute for the word “similarity.” A partiallycomplementary sequence that at least partially inhibits an identicalsequence from hybridizing to a target nucleic acid is referred to as“substantially similar.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization, and the like) under conditions of reduced stringency. Asubstantially similar sequence or hybridization probe will compete forand inhibit the binding of a completely similar (identical) sequence tothe target sequence under conditions of reduced stringency. This is notto say that conditions of reduced stringency are such that non-specificbinding is permitted, as reduced stringency conditions require that thebinding of two sequences to one another be a specific (i.e., aselective) interaction. The absence of non-specific binding may betested by the use of a second target sequence which lacks even a partialdegree of complementarity (e.g., less than about 30% similarity oridentity). In the absence of non-specific binding, the substantiallysimilar sequence or probe will not hybridize to the secondnon-complementary target sequence.

[0044] The phrases “percent identity” or “% identity” refer to thepercentage of sequence similarity found in a comparison of two or moreamino acid or nucleic acid sequences. Percent identity can be determinedelectronically, e.g., by using the MEGALIGN program (DNASTAR) whichcreates alignments between two or more sequences according to methodsselected by the user, e.g., the clustal method. (See, e.g., Higgins, D.G. and P. M. Sharp (1988) Gene 73:237-244.) The clustal algorithm groupssequences into clusters by examining the distances between all pairs.The clusters are aligned pairwise and then in groups. The percentagesimilarity between two amino acid sequences, e.g., sequence A andsequence B, is calculated by dividing the length of sequence A, minusthe number of gap residues in sequence A, minus the number of gapresidues in sequence B, into the sum of the residue matches betweensequence A and sequence B, times one hundred. Gaps of low or of nosimilarity between the two amino acid sequences are not included indetermining percentage similarity. Percent identity between nucleic acidsequences can also be counted or calculated by other methods known inthe art, e.g., the Jotun Hein method. (See, e.g., Hein, J. (1990)Methods Enzymol. 183:626-645.) Identity between sequences can also bedetermined by other methods known in the art, e.g., by varyinghybridization conditions.

[0045] “Human artificial chromosomes” (HACs) are linear microchromosomeswhich may contain DNA sequences of about 6 kb to 10 Mb in size, andwhich contain all of the elements required for stable mitotic chromosomesegregation and maintenance.

[0046] The term “humanized antibody” refers to antibody molecules inwhich the amino acid sequence in the non-antigen binding regions hasbeen altered so that the antibody more closely resembles a humanantibody, and still retains its original binding ability.

[0047] “Hybridization” refers to any process by which a strand ofnucleic acid binds with a complementary strand through base pairing.

[0048] The term “hybridization complex” refers to a complex formedbetween two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary bases. A hybridization complex maybe formed in solution (e.g., C₀t or R₀t analysis) or formed between onenucleic acid sequence present in solution and another nucleic acidsequence immobilized on a solid support (e.g., paper, membranes,filters, chips, pins or glass slides, or any other appropriate substrateto which cells or their nucleic acids have been fixed).

[0049] The words “insertion” or “addition” refer to changes in an aminoacid or nucleotide sequence resulting in the addition of one or moreamino acid residues or nucleotides, respectively, to the sequence foundin the naturally occurring molecule.

[0050] “Immune response” can refer to conditions associated withinflammation, trauma, immune disorders, or infectious or geneticdisease, etc. These conditions can be characterized by expression ofvarious factors, e.g., cytokines, chemokines, and other signalingmolecules, which may affect cellular and systemic defense systems.

[0051] The term “microarray” refers to an arrangement of distinctpolynucleotides on a substrate.

[0052] The terms “element” or “array element” in a microarray context,refer to hybridizable polynucleotides arranged on the surface of asubstrate.

[0053] The term “modulate” refers to a change in the activity of hCBP.For example, modulation may cause an increase or a decrease in proteinactivity, binding characteristics, or any other biological, functional,or immunological properties of hCBP.

[0054] The phrases “nucleic acid” or “nucleic acid sequence” refer to anucleotide, oligonucleotide, polynucleotide, or any fragment thereof.These phrases also refer to DNA or RNA of genomic or synthetic originwhich may be single-stranded or double-stranded and may represent thesense or the antisense strand, to peptide nucleic acid (PNA), or to anyDNA-like or RNA-like material. In this context, “fragments” refers tothose nucleic acid sequences which, when translated, would producepolypeptides retaining some functional characteristic, e.g.,antigenicity, or structural domain characteristic, e.g., ATP-bindingsite, of the full-length polypeptide.

[0055] The terms “operably associated” or “operably linked” refer tofunctionally related nucleic acid sequences. A promoter is operablyassociated or operably linked with a coding sequence if the promotercontrols the translation of the encoded polypeptide. While operablyassociated or operably linked nucleic acid sequences can be contiguousand in the same reading frame, certain genetic elements, e.g., repressorgenes, are not contiguously linked to the sequence encoding thepolypeptide but still bind to operator sequences that control expressionof the polypeptide.

[0056] The term “oligonucleotide” refers to a nucleic acid sequence ofat least about 6 nucleotides to 60 nucleotides, preferably about 15 to30 nucleotides, and most preferably about 20 to 25 nucleotides, whichcan be used in PCR amplification or in a hybridization assay ormicroarray. “Oligonucleotide” is substantially equivalent to the terms“amplimer,” “primer,” “oligomer,” and “probe,” as these terms arecommonly defined in the art.

[0057] “Peptide nucleic acid” (PNA) refers to an antisense molecule oranti-gene agent which comprises an oligonucleotide of at least about 5nucleotides in length linked to a peptide backbone of amino acidresidues ending in lysine. The terminal lysine confers solubility to thecomposition. PNAs preferentially bind complementary single stranded DNAor RNA and stop transcript elongation, and may be pegylated to extendtheir lifespan in the cell.

[0058] The term “sample” is used in its broadest sense. A samplesuspected of containing nucleic acids encoding hCBP, or fragmentsthereof, or hCBP itself, may comprise a bodily fluid; an extract from acell, chromosome, organelle, or membrane isolated from a cell; a cell;genomic DNA, RNA, or cDNA, in solution or bound to a substrate; atissue; a tissue print; etc.

[0059] The terms “specific binding” or “specifically binding” refer tothat interaction between a protein or peptide and an agonist, anantibody, or an antagonist. The interaction is dependent upon thepresence of a particular structure of the protein, e.g., the antigenicdeterminant or epitope, recognized by the binding molecule. For example,if an antibody is specific for epitope “A,” the presence of apolypeptide containing the epitope A, or the presence of free unlabeledA, in a reaction containing free labeled A and the antibody will reducethe amount of labeled A that binds to the antibody.

[0060] The term “stringent conditions” refers to conditions which permithybridization between polynucleotides and the claimed polynucleotides.Stringent conditions can be defined by salt concentration, theconcentration of organic solvent, e.g., formamide, temperature, andother conditions well known in the art. In particular, stringency can beincreased by reducing the concentration of salt, increasing theconcentration of formamide, or raising the hybridization temperature.

[0061] The term “substantially purified” refers to nucleic acid or aminoacid sequences that are removed from their natural environment and areisolated or separated, and are at least about 60% free, preferably about75% free, and most preferably about 90% free from other components withwhich they are naturally associated.

[0062] A “substitution” refers to the replacement of one or more aminoacids or nucleotides by different amino acids or nucleotides,respectively.

[0063] “Substrate” refers to any suitable rigid or semi-rigid supportincluding membranes, filters, chips, slides, wafers, fibers, magnetic ornonmagnetic beads, gels, tubing, plates, polymers, microparticles andcapillaries. The substrate can have a variety of surface forms, such aswells, trenches, pins, channels and pores, to which polynucleotides orpolypeptides are bound.

[0064] “Transformation” describes a process by which exogenous DNAenters and changes a recipient cell. Transformation may occur undernatural or artificial conditions according to various methods well knownin the art, and may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method for transformation is selected based on the type ofhost cell being transformed and may include, but is not limited to,viral infection, electroporation, heat shock, lipofection, and particlebombardment. The term “transformed” cells includes stably transformedcells in which the inserted DNA is capable of replication either as anautonomously replicating plasmid or as part of the host chromosome, aswell as transiently transformed cells which express the inserted DNA orRNA for limited periods of time.

[0065] A “variant” of hCBP polypeptides refers to an amino acid sequencethat is altered by one or more amino acid residues. The variant may have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties (e.g., replacement of leucine withisoleucine). More rarely, a variant may have “nonconservative” changes(e.g., replacement of glycine with tryptophan). Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, LASERGENE software (DNASTAR).

[0066] The term “variant,” when used in the context of a polynucleotidesequence, may encompass a polynucleotide sequence related to hCBP. Thisdefinition may also include, for example, “allelic” (as defined above),“splice,” “species,” or “polymorphic” variants. A splice variant mayhave significant identity to a reference molecule, but will generallyhave a greater or lesser number of polynucleotides due to alternatesplicing of exons during mRNA processing. The corresponding polypeptidemay possess additional functional domains or an absence of domains.Species variants are polynucleotide sequences that vary from one speciesto another. The resulting polypeptides generally will have significantamino acid identity relative to each other. A polymorphic variant is avariation in the polynucleotide sequence of a particular gene betweenindividuals of a given species. Polymorphic variants also may encompass“single nucleotide polymorphisms” (SNPs) in which the polynucleotidesequence varies by one base. The presence of SNPs may be indicative of,for example, a certain population, a disease state, or a propensity fora disease state.

[0067] The Invention

[0068] The invention is based on the discovery of a new human calciumbinding protein (hCBP), the polynucleotides encoding hCBP, and the useof these compositions for the diagnosis, treatment, or prevention ofcancer, reproductive disorders, immune disorders, and developmentaldisorders.

[0069] Nucleic acids encoding the hCBP of the present invention wereidentified in Incyte Clone 3734805H1 from the coronary artery smoothmuscle cDNA library (SMCCNOS01) using a computer search for nucleotideand/or amino acid sequence alignments. A consensus sequence, SEQ IDNO:2, was derived from the following overlapping and/or extended nucleicacid sequences: Incyte Clones 3734805H1 (SMCCNOS01), 3765972F6(BRSTNOT24), 2260704X21C2 (UTRSNOT02), 2497543T6 (ADRETUT05), andshotgun sequence SBIA06584D1.

[0070] In one embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A,1B, 1C and 1D. hCBP is 337 amino acids in length and has one potentialcAMP- and cGMP-dependent protein kinase phosphorylation site at residueS228, five potential casein kinase II phosphorylation sites at residuesS175, T186, T208, S250, and T313, and five potential protein kinase Cphosphorylation sites at S10, T35, T186, T224, and S228. PRINTS analysisreveals a putative Huntingtin signature in hCBP within the region fromabout residues T224 through A243. The gene coding for Huntingtin, aprotein associated with Huntington's disease contains a fragment that isunstable on Huntington's disease chromosomes. As shown in FIGS. 2A and2B, hCBP has chemical and structural similarity with MO25 (GI 262934;SEQ ID NO:3), DMO25 (GI 1794137; SEQ ID NO:4), and yeast-like CBP (GI1255838; SEQ ID NO:5). In particular, hCBP and MO25 share 80% identity,hCBP and DMO25 share 64% identity, and hCBP and yeast-like CBP share 60%identity. The Huntingtin signature found in hCBP is conserved in allthree reference proteins, MO25, DMO25, and yeast-like CBP. In addition,the potential cAMP- and cGMP-dependent protein kinase phosphorylationsite at residue S228, the potential casein kinase II phosphorylationsites at residues T186 and S250, and the potential protein kinase Cphosphorylation sites at S10, T186, T224, and S228 of hCBP are conservedin MO25, DMO25, and yeast-like CBP. A fragment of SEQ ID NO:2 from aboutnucleotide 157 to about nucleotide 204 is useful in hybridization oramplification technologies to identify SEQ ID NO:2 and to distinguishbetween SEQ ID NO:2 and a related sequence. Northern analysis shows theexpression of this sequence in various libraries, at least 44% of whichare associated with cancer and at least 22% of which are associated withinflammation and the immune response. Of particular note is theexpression of hCBP in reproductive tissue.

[0071] The invention also encompasses hCBP variants. A preferred hCBPvariant is one which has at least about 80%, more preferably at leastabout 90%, and most preferably at least about 95% amino acid sequenceidentity to the hCBP amino acid sequence, and which contains at leastone functional or structural characteristic of hCBP.

[0072] The invention also encompasses polynucleotides which encode hCBP.In a particular embodiment, the invention encompasses a polynucleotidesequence comprising the sequence of SEQ ID NO:2, which encodes hCBP.

[0073] The invention also encompasses a variant of a polynucleotidesequence encoding hCBP. In particular, such a variant polynucleotidesequence will have at least about 70%, more preferably at least about85%, and most preferably at least about 95% polynucleotide sequenceidentity to the polynucleotide sequence encoding hCBP. A particularaspect of the invention encompasses a variant of SEQ ID NO:2 which hasat least about 70%, more preferably at least about 85%, and mostpreferably at least about 95% polynucleotide sequence identity to SEQ IDNO:2. Any one of the polynucleotide variants described above can encodean amino acid sequence which contains at least one functional orstructural characteristic of hCBP.

[0074] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude ofpolynucleotide sequences encoding hCBP, some bearing minimal similarityto the polynucleotide sequences of any known and naturally occurringgene, may be produced. Thus, the invention contemplates each and everypossible variation of polynucleotide sequence that could be made byselecting combinations based on possible codon choices. Thesecombinations are made in accordance with the standard triplet geneticcode as applied to the polynucleotide sequence of naturally occurringhCBP, and all such variations are to be considered as being specificallydisclosed.

[0075] Although nucleotide sequences which encode hCBP and its variantsare preferably capable of hybridizing to the nucleotide sequence of thenaturally occurring hCBP under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding hCBP or its derivatives possessing a substantially differentcodon usage, e.g., inclusion of non-naturally occurring codons. Codonsmay be selected to increase the rate at which expression of the peptideoccurs in a particular prokaryotic or eukaryotic host in accordance withthe frequency with which particular codons are utilized by the host.Other reasons for substantially altering the nucleotide sequenceencoding hCBP and its derivatives without altering the encoded aminoacid sequences include the production of RNA transcripts having moredesirable properties, such as a greater half-life, than transcriptsproduced from the naturally occurring sequence.

[0076] The invention also encompasses production of DNA sequences whichencode hCBP and hCBP derivatives, or fragments thereof, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents well known in the art. Moreover, syntheticchemistry may be used to introduce mutations into a sequence encodinghCBP or any fragment thereof.

[0077] Also encompassed by the invention are polynucleotide sequencesthat are capable of hybridizing to the claimed polynucleotide sequences,and, in particular, to those shown in SEQ ID NO:2, or to a fragment ofSEQ ID NO:2, under various conditions of stringency. (See, e.g., Wahl,G. M. and S. L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods Enzymol. 152:507-511.) For example, stringent saltconcentration will ordinarily be less than about 750 mM NaCl and 75 mMtrisodium citrate, preferably less than about 500 mM NaCl and 50 mMtrisodium citrate, and most preferably less than about 250 mM NaCl and25 mM trisodium citrate. Low stringency hybridization can be obtained inthe absence of organic solvent, e.g., formamide, while high stringencyhybridization can be obtained in the presence of at least about 35%formamide, and most preferably at least about 50% formamide. Stringenttemperature conditions will ordinarily include temperatures of at leastabout 30° C., more preferably of at least about 37° C., and mostpreferably of at least about 42° C. Varying additional parameters, suchas hybridization time, the concentration of detergent, e.g., sodiumdodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA,are well known to those skilled in the art. Various levels of stringencyare accomplished by combining these various conditions as needed. In apreferred embodiment, hybridization will occur at 30° C. in 750 mM NaCl,75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment,hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodiumcitrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA(ssDNA). In a most preferred embodiment, hybridization will occur at 42°C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and200 μg/ml ssDNA. Useful variations on these conditions will be readilyapparent to those skilled in the art.

[0078] The washing steps which follow hybridization can also vary instringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude temperature of at least about 25° C., more preferably of atleast about 42° C., and most preferably of at least about 68° C. In apreferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a most preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art.

[0079] Methods for DNA sequencing are well known in the art and may beused to practice any of the embodiments of the invention. The methodsmay employ such enzymes as the Klenow fragment of DNA polymerase I,SEQUENASE (US Biochemical, Cleveland Ohio), Taq polymerase(Perkin-Elmer), thermostable T7 polymerase (Amersham Pharmacia Biotech,Piscataway N.J.), or combinations of polymerases and proofreadingexonucleases such as those found in the ELONGASE amplification system(Life Technologies, Gaithersburg Md.). Preferably, sequence preparationis automated with machines such as the Robbins Hydra microdispenser(Robbins Scientific, Sunnyvale Calif.), MICROLAB 2200 (Hamilton, RenoNev.), Peltier thermal cycler 200 (PTC200; MJ Research, Watertown Mass.)and the ABI CATALYST 800 (Perkin-Elmer). Sequencing is then carried outusing either ABI 373 or 377 DNA sequencing systems (Perkin-Elmer) or theMEGABACE 1000 DNA sequencing system (Molecular Dynamics, SunnyvaleCalif.). The resulting sequences are analyzed using a variety ofalgorithms which are well known in the art. (See, e.g., Ausubel, F. M.(1997) Short Protocols in Molecular Biology, John Wiley & Sons, New YorkN.Y., unit 7.7; Meyers, R. A. (1995) Molecular Biology andBiotechnology, Wiley VCH, New York N.Y., pp. 856-853.)

[0080] The nucleic acid sequences encoding hCBP may be extendedutilizing a partial nucleotide sequence and employing various PCR-basedmethods known in the art to detect upstream sequences, such as promotersand regulatory elements. For example, one method which may be employed,restriction-site PCR, uses universal and nested primers to amplifyunknown sequence from genomic DNA within a cloning vector. (See, e.g.,Sarkar, G. (1993) PCR Methods Applic. 2:318-322.) Another method,inverse PCR, uses primers that extend in divergent directions to amplifyunknown sequence from a circularized template. The template is derivedfrom restriction fragments comprising a known genomic locus andsurrounding sequences. (See, e.g., Triglia, T. et al. (1988) NucleicAcids Res. 16:8186.) A third method, capture PCR, involves PCRamplification of DNA fragments adjacent to known sequences in human andyeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al.(1991) PCR Methods Applic. 1:111-119.) In this method, multiplerestriction enzyme digestions and ligations may be used to insert anengineered double-stranded sequence into a region of unknown sequencebefore performing PCR. Other methods which may be used to retrieveunknown sequences are known in the art. (See, e.g., Parker, J. D. et al.(1991) Nucleic Acids Res. 19:3055-306). Additionally, one may use PCR,nested primers, and PROMOTERFINDER libraries (Clontech, Palo AltoCalif.) to walk genomic DNA. This procedure avoids the need to screenlibraries and is useful in finding intron/exon junctions. For allPCR-based methods, primers may be designed using commercially availablesoftware, such as OLIGO 4.06 primer analysis software (NationalBiosciences, Plymouth Minn.) or another appropriate program, to be about22 to 30 nucleotides in length, to have a GC content of about 50% ormore, and to anneal to the template at temperatures of about 68° C. to72° C.

[0081] When screening for full-length cDNAs, it is preferable to uselibraries that have been size-selected to include larger cDNAs. Inaddition, random-primed libraries, which often include sequencescontaining the 5′ regions of genes, are preferable for situations inwhich an oligo d(T) library does not yield a full-length cDNA. Genomiclibraries may be useful for extension of sequence into 5′non-transcribed regulatory regions.

[0082] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different nucleotide-specific, laser-stimulated fluorescent dyes,and a charge coupled device camera for detection of the emittedwavelengths. Output/light intensity may be converted to electricalsignal using appropriate software (e.g., GENOTYPER and SEQUENCENAVIGATOR, Perkin-Elmer), and the entire process from loading of samplesto computer analysis and electronic data display may be computercontrolled. Capillary electrophoresis is especially preferable forsequencing small DNA fragments which may be present in limited amountsin a particular sample.

[0083] In another embodiment of the invention, polynucleotide sequencesor fragments thereof which encode hCBP may be cloned in recombinant DNAmolecules that direct expression of hCBP, or fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and used to express hCBP.

[0084] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterhCBP-encoding sequences for a variety of purposes including, but notlimited to, modification of the cloning, processing, and/or expressionof the gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example,oligonucleotide-mediated site-directed mutagenesis may be used tointroduce mutations that create new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, and so forth.

[0085] In another embodiment, sequences encoding hCBP may besynthesized, in whole or in part, using chemical methods well known inthe art. (See, e.g., Caruthers, M. H. et al. (1980) Nucl. Acids Res.Symp. Ser. 215-223, and Horn, T. et al. (1980) Nucl. Acids Res. Symp.Ser. 225-232.) Alternatively, hCBP itself or a fragment thereof may besynthesized using chemical methods. For example, peptide synthesis canbe performed using various solid-phase techniques. (See, e.g., Roberge,J. Y. et al. (1995) Science 269:202-204.) Automated synthesis may beachieved using the ABI 431A peptide synthesizer (Perkin-Elmer).Additionally, the amino acid sequence of hCBP, or any part thereof, maybe altered during direct synthesis and/or combined with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

[0086] The peptide may be substantially purified by preparative highperformance liquid chromatography. (See, e.g, Chiez, R. M. and F. Z.Regnier (1990) Methods Enzymol. 182:392-421.) The composition of thesynthetic peptides may be confirmed by amino acid analysis or bysequencing. (See, e.g., Creighton, T. (1984) Proteins, Structures andMolecular Properties, W H Freeman, New York N.Y.)

[0087] In order to express a biologically active hCBP, the nucleotidesequences encoding hCBP or derivatives thereof may be inserted into anappropriate expression vector, i.e., a vector which contains thenecessary elements for transcriptional and translational control of theinserted coding sequence in a suitable host. These elements includeregulatory sequences, such as enhancers, constitutive and induciblepromoters, and 5′ and 3′ untranslated regions in the vector and inpolynucleotide sequences encoding hCBP. Such elements may vary in theirstrength and specificity. Specific initiation signals may also be usedto achieve more efficient translation of sequences encoding hCBP. Suchsignals include the ATG initiation codon and adjacent sequences, e.g.the Kozak sequence. In cases where sequences encoding hCBP and itsinitiation codon and upstream regulatory sequences are inserted into theappropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including an in-frame ATG initiation codonshould be provided by the vector. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers appropriate for the particular host cell system used. (See,e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)

[0088] Methods which are well known to those skilled in the art may beused to construct expression vectors containing sequences encoding hCBPand appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, e.g., Sambrook, J.et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Plainview N.Y., ch. 4, 8, and 16-17; Ausubel, F. M. et al. (1995)Current Protocols in Molecular Biology, John Wiley & Sons, New YorkN.Y., ch. 9, 13, and 16.)

[0089] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding hCBP. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith viral expression vectors (e.g., baculovirus); plant cell systemstransformed with viral expression vectors (e.g., cauliflower mosaicvirus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

[0090] In bacterial systems, a number of cloning and expression vectorsmay be selected depending upon the use intended for polynucleotidesequences encoding hCBP. For example, routine cloning, subcloning, andpropagation of polynucleotide sequences encoding hCBP can be achievedusing a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene,La Jolla Calif.) or PSPORT1 plasmid (Life Technologies). Ligation ofsequences encoding hCBP into the vector's multiple cloning site disruptsthe lacZ gene, allowing a colorimetric screening procedure foridentification of transformed bacteria containing recombinant molecules.In addition, these vectors may be useful for in vitro transcription,dideoxy sequencing, single strand rescue with helper phage, and creationof nested deletions in the cloned sequence. (See, e.g., Van Heeke, G.and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) When largequantities of hCBP are needed, e.g. for the production of antibodies,vectors which direct high level expression of hCBP may be used. Forexample, vectors containing the strong, inducible T5 or T7 bacteriophagepromoter may be used.

[0091] Yeast expression systems may be used for production of hCBP. Anumber of vectors containing constitutive or inducible promoters, suchas alpha factor, alcohol oxidase, and PGH promoters, may be used in theyeast Saccharomvces cerevisiae or Pichia pastoris. In addition, suchvectors direct either the secretion or intracellular retention ofexpressed proteins and enable integration of foreign sequences into thehost genome for stable propagation. (See, e.g., Ausubel, 1995, supra;Grant et al. (1987) Methods Enzymol. 153:516-54; and Scorer, C. A. etal. (1994) Bio/Technology 12:181-184.)

[0092] Plant systems may also be used for expression of hCBP.Transcription of sequences encoding hCBP may be driven by viralpromoters, e.g., the 35S and 19S promoters of CaMV used alone or incombination with the omega leader sequence from TMV (Takamatsu, N.(1987) EMBO J. 6:307-311). Alternatively, plant promoters such as thesmall subunit of RUBISCO or heat shock promoters may be used. (See,e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al.(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl.Cell Differ. 17:85-105.) These constructs can be introduced into plantcells by direct DNA transformation or pathogen-mediated transfection.(See, e.g., The McGraw Hill Yearbook of Science and Technology (1992)McGraw Hill, New York N.Y., pp. 191-196.)

[0093] In mammalian cells, a number of viral-based expression systemsmay be utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding hCBP may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain infective virus whichexpresses hCBP in host cells. (See, e.g., Logan, J. and T. Shenk (1984)Proc. Natl. Acad. Sci. 81:3655-3659.) In addition, transcriptionenhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used toincrease expression in mammalian host cells. SV40 or EBV-based vectorsmay also be used for high-level protein expression.

[0094] Human artificial chromosomes (HACs) may also be employed todeliver larger fragments of DNA than can be contained in and expressedfrom a plasmid. HACs of about 6 kb to 10 Mb are constructed anddelivered via conventional delivery methods (liposomes, polycationicamino polymers, or vesicles) for therapeutic purposes. (See, e.g.,Harrington, J. J. et al. (1997) Nat. Genet. 15:345-355.) For long termproduction of recombinant proteins in mammalian systems, stableexpression of hCBP in cell lines is preferred. For example, sequencesencoding hCBP can be transformed into cell lines using expressionvectors which may contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. Following the introduction of the vector, cells may beallowed to grow for about 1 to 2 days in enriched media before beingswitched to selective media. The purpose of the selectable marker is toconfer resistance to a selective agent, and its presence allows growthand recovery of cells which successfully express the introducedsequences. Resistant clones of stably transformed cells may bepropagated using tissue culture techniques appropriate to the cell type.

[0095] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase and adeninephosphoribosyltransferase genes, for use in tk⁻ or apr⁻ cells,respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11:223-232;Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabolite,antibiotic, or herbicide resistance can be used as the basis forselection. For example, dhfr confers resistance to methotrexate; neoconfers resistance to the aminoglycosides, neomycin and G-418; and alsor pat confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively. (See, e.g., Wigler, M. et al. (1980)Proc. Natl. Acad. Sci. 77:3567-3570; Colbere-Garapin, F. et al. (1981)J. Mol. Biol. 150:1-14.) Additional selectable genes have beendescribed, e.g., trpB and hisD, which alter cellular requirements formetabolites. (See, e.g., Hartman, S. C. and R. C. Mulligan (1988) Proc.Natl. Acad. Sci. 85:8047-8051.) Visible markers, e.g., anthocyanins,green fluorescent proteins (GFP; Clontech), B glucuronidase and itssubstrate β-glucuronide, or luciferase and its substrate luciferin maybe used. These markers can be used not only to identify transformants,but also to quantify the amount of transient or stable proteinexpression attributable to a specific vector system. (See, e.g., Rhodes,C. A. (1995) Methods Mol. Biol. 55:121-131.)

[0096] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, the presence and expressionof the gene may need to be confirmed. For example, if the sequenceencoding hCBP is inserted within a marker gene sequence, transformedcells containing sequences encoding hCBP can be identified by theabsence of marker gene function. Alternatively, a marker gene can beplaced in tandem with a sequence encoding hCBP under the control of asingle promoter. Expression of the marker gene in response to inductionor selection usually indicates expression of the tandem gene as well.

[0097] In general, host cells that contain the nucleic acid sequenceencoding hCBP and that express hCBP may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCRamplification, and protein bioassay or immunoassay techniques whichinclude membrane, solution, or chip based technologies for the detectionand/or quantification of nucleic acid or protein sequences.

[0098] Immunological methods for detecting and measuring the expressionof hCBP using either specific polyclonal or monoclonal antibodies areknown in the art. Examples of such techniques include enzyme-linkedimmunosorbent assays (ELISAs), radioimmunoassays (RIAs), andfluorescence activated cell sorting (FACS). A two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes on hCBP is preferred, but a competitive bindingassay may be employed. These and other assays are well known in the art.(See, e.g., Hampton, R. et al. (1990) Serological Methods, a LaboratoryManual, APS Press, St Paul Minn., Sect. IV; Coligan, J. E. et al. (1997)Current Protocols in Immunology, Greene Pub. Associates andWiley-Interscience, New York N.Y.; and Pound, J. D. (1998)Immunochemical Protocols, Humana Press, Totowa N.J.).

[0099] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encoding hCBPinclude oligolabeling, nick translation, end-labeling, or PCRamplification using a labeled nucleotide. Alternatively, the sequencesencoding hCBP, or any fragments thereof, may be cloned into a vector forthe production of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits, such as those provided byAmersham Pharmacia Biotech, Promega (Madison Wis.), and US Biochemical.Suitable reporter molecules or labels which may be used for ease ofdetection include radionuclides, enzymes, fluorescent, chemiluminescent,or chromogenic agents, as well as substrates, cofactors, inhibitors,magnetic particles, and the like.

[0100] Host cells transformed with nucleotide sequences encoding hCBPmay be cultured under conditions suitable for the expression andrecovery of the protein from cell culture. The protein produced by atransformed cell may be secreted or retained intracellularly dependingon the sequence and/or the vector used. As will be understood by thoseof skill in the art, expression vectors containing polynucleotides whichencode hCBP may be designed to contain signal sequences which directsecretion of hCBP through a prokaryotic or eukaryotic cell membrane.

[0101] In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to specify protein targeting, folding, and/oractivity. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and W138), are available from the American TypeCulture Collection (ATCC, Bethesda Md.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

[0102] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding hCBP may be ligated to aheterologous sequence resulting in translation of a fusion protein inany of the aforementioned host systems. For example, a chimeric hCBPprotein containing a heterologous moiety that can be recognized by acommercially available antibody may facilitate the screening of peptidelibraries for inhibitors of hCBP activity. Heterologous protein andpeptide moieties may also facilitate purification of fusion proteinsusing commercially available affinity matrices. Such moieties include,but are not limited to, glutathione S-transferase (GST), maltose bindingprotein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP),6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and6-His enable purification of their cognate fusion proteins onimmobilized glutathione, maltose, phenylarsine oxide, calmodulin, andmetal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA)enable immunoaffinity purification of fusion proteins using commerciallyavailable monoclonal and polyclonal antibodies that specificallyrecognize these epitope tags. A fusion protein may also be engineered tocontain a proteolytic cleavage site located between the hCBP encodingsequence and the heterologous protein sequence, so that hCBP may becleaved away from the heterologous moiety following purification.Methods for fusion protein expression and purification are discussed inAusubel (1995, supra, ch 10). A variety of commercially available kitsmay also be used to facilitate expression and purification of fusionproteins.

[0103] In a further embodiment of the invention, synthesis ofradiolabeled hCBP may be achieved in vitro using the TNT rabbitreticulocyte lysate or wheat germ extract systems (Promega). Thesesystems couple transcription and translation of protein-coding sequencesoperably associated with the T7, T3, or SP6 promoters. Translation takesplace in the presence of a radiolabeled amino acid precursor, preferably³⁵S-methionine.

[0104] Fragments of hCBP may be produced not only by recombinantproduction, but also by direct peptide synthesis using solid-phasetechniques. (See, e.g., Creighton, supra pp. 55-60.) Protein synthesismay be performed by manual techniques or by automation. Automatedsynthesis may be achieved, for example, using the ABI 431A PeptideSynthesizer (Perkin-Elmer). Various fragments of hCBP may be synthesizedseparately and then combined to produce the full length molecule.

[0105] Therapeutics

[0106] Chemical and structural similarity, e.g., in the context ofsequences and motifs, exists between regions of hCBP, MO25 (GI 262934),DMO25 (GI 1794137), and yeast-like CBP (GI 1255838). In addition, theexpression of hCBP is closely associated with cancer, reproductivetissues, inflammation and the immune response. Therefore, hCBP appearsto play a role in cancer, reproductive disorders, immune disorders, anddevelopmental disorders. In the treatment of cancer, reproductivedisorders, immune disorders, and developmental disorders associated withincreased hCBP expression or activity, it is desirable to decrease theexpression or activity of hCBP. In the treatment of the above conditionsassociated with decreased hCBP expression or activity, it is desirableto increase the expression or activity of hCBP.

[0107] Therefore, in one embodiment, hCBP or a fragment or derivativethereof may be administered to a subject to treat or prevent a disorderassociated with decreased expression or activity of hCBP. Examples ofsuch disorders include, but are not limited to, a cancer, such asadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; a reproductive disordersuch as disorders of prolactin production, infertility, including tubaldisease, ovulatory defects, and endometriosis, disruptions of theestrous cycle, disruptions of the menstrual cycle, polycystic ovarysyndrome, ovarian hyperstimulation syndrome, endometrial and ovariantumors, uterine fibroids, autoimmune disorders, ectopic pregnancies, andteratogenesis, cancer of the breast, fibrocystic breast disease, andgalactorrhea, disruptions of spermatogenesis, abnormal sperm physiology,cancer of the testis, cancer of the prostate, benign prostatichyperplasia, prostatitis, Peyronie's disease, impotence, carcinoma ofthe male breast, and gynecomastia; a developmental disorder, such asrenal tubular acidosis, anemia, Cushing's syndrome, achondroplasticdwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadaldysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinaryabnormalities, and mental retardation), Smith-Magenis syndrome,myelodysplastic syndrome, hereditary mucoepithelial dysplasia,hereditary keratodermas, hereditary neuropathies such asCharcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism,hydrocephalus, seizure disorders such as Syndenham's chorea and cerebralpalsy, spina bifida, anencephaly, craniorachischisis, congenitalglaucoma, cataract, and sensorineural hearing loss; and an immunedisorder, such as acquired immunodeficiency syndrome (AIDS), Addison'sdisease, adult respiratory distress syndrome, allergies, ankylosingspondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmunehemolytic anemia, autoimmune thyroiditis, autoimmunepolyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED),bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopicdermatitis, dermatomyositis, diabetes mellitus, emphysema, episodiclymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythemanodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome,gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,irritable bowel syndrome, multiple sclerosis, myasthenia gravis,myocardial or pericardial inflammation, osteoarthritis, osteoporosis,pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoidarthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis,systemic lupus erythematosus, systemic sclerosis, thrombocytopenicpurpura, ulcerative colitis, uveitis, Werner syndrome, complications ofcancer, hemodialysis, and extracorporeal circulation, viral, bacterial,fungal, parasitic, protozoal, and helminthic infections, and trauma.

[0108] In another embodiment, a vector capable of expressing hCBP or afragment or derivative thereof may be administered to a subject to treator prevent a disorder associated with decreased expression or activityof hCBP including, but not limited to, those described above.

[0109] In a further embodiment, a pharmaceutical composition comprisinga substantially purified hCBP in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent a disorder associated with decreased expression or activity ofhCBP including, but not limited to, those provided above.

[0110] In still another embodiment, an agonist which modulates theactivity of hCBP may be administered to a subject to treat or prevent adisorder associated with decreased expression or activity of hCBPincluding, but not limited to, those listed above.

[0111] In a further embodiment, an antagonist of hCBP may beadministered to a subject to treat or prevent a disorder associated withincreased expression or activity of HCBP. Such disorders may include,but are not limited to, those discussed above. In one aspect, anantibody which specifically binds hCBP may be used directly as anantagonist or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express hCBP.

[0112] In an additional embodiment, a vector expressing the complementof the polynucleotide encoding hCBP may be administered to a subject totreat or prevent a disorder associated with increased expression oractivity of hCBP including, but not limited to, those described above.

[0113] In other embodiments, any of the proteins, antagonists,antibodies, agonists, complementary sequences, or vectors of theinvention may be administered in combination with other appropriatetherapeutic agents. Selection of the appropriate agents for use incombination therapy may be made by one of ordinary skill in the art,according to conventional pharmaceutical principles. The combination oftherapeutic agents may act synergistically to effect the treatment orprevention of the various disorders described above. Using thisapproach, one may be able to achieve therapeutic efficacy with lowerdosages of each agent, thus reducing the potential for adverse sideeffects.

[0114] An antagonist of hCBP may be produced using methods which aregenerally known in the art. In particular, purified hCBP may be used toproduce antibodies or to screen libraries of pharmaceutical agents toidentify those which specifically bind hCBP. Antibodies to hCBP may alsobe generated using methods that are well known in the art. Suchantibodies may include, but are not limited to, polyclonal, monoclonal,chimeric, and single chain antibodies, Fab fragments, and fragmentsproduced by a Fab expression library. Neutralizing antibodies (i.e.,those which inhibit dimer formation) are especially preferred fortherapeutic use.

[0115] For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others may be immunized by injectionwith hCBP or with any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable.

[0116] It is preferred that the oligopeptides, peptides, or fragmentsused to induce antibodies to hCBP have an amino acid sequence consistingof at least about 5 amino acids, and, more preferably, of at least about10 amino acids. It is also preferable that these oligopeptides,peptides, or fragments are identical to a portion of the amino acidsequence of the natural protein and contain the entire amino acidsequence of a small, naturally occurring molecule. Short stretches ofhCBP amino acids may be fused with those of another protein, such asKLH, and antibodies to the chimeric molecule may be produced.

[0117] Monoclonal antibodies to hCBP may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique. (See, e.g., Kohler, G. et al. (1975) Nature256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42;Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; and Cole,S. P. et al. (1984) Mol. Cell Biol. 62:109-120.)

[0118] In addition, techniques developed for the production of “chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used. (See, e.g., Morrison, S. L. et al.(1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al.(1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature314:452-454.) Alternatively, techniques described for the production ofsingle chain antibodies may be adapted, using methods known in the art,to produce hCBP-specific single chain antibodies. Antibodies withrelated specificity, but of distinct idiotypic composition, may begenerated by chain shuffling from random combinatorial immunoglobulinlibraries. (See, e.g., Burton D. R. (1991) Proc. Natl. Acad. Sci.88:10134-10137.)

[0119] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening immunoglobulin libraries orpanels of highly specific binding reagents as disclosed in theliterature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.86: 3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)

[0120] Antibody fragments which contain specific binding sites for hCBPmay also be generated. For example, such fragments include, but are notlimited to, F(ab′)2 fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab′)2 fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity. (See, e.g., Huse,W. D. et al. (1989) Science 246:1275-1281.) Various immunoassays may beused for screening to identify antibodies having the desiredspecificity. Numerous protocols for competitive binding orimmunoradiometric assays using either polyclonal or monoclonalantibodies with established specificities are well known in the art.Such immunoassays typically involve the measurement of complex formationbetween hCBP and its specific antibody. A two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering hCBP epitopes is preferred, but a competitive bindingassay may also be employed (Pound, supra).

[0121] Various methods such as Scatchard analysis in conjunction withradioimmunoassay techniques may be used to assess the affinity ofantibodies for hCBP. Affinity is expressed as an association constant,K_(a), which is defined as the molar concentration of hCBP-antibodycomplex divided by the molar concentrations of free antigen and freeantibody under equilibrium conditions. The K_(a) determined for apreparation of polyclonal antibodies, which are heterogeneous in theiraffinities for multiple hCBP epitopes, represents the average affinity,or avidity, of the antibodies for hCBP. The K_(a) determined for apreparation of monoclonal antibodies, which are monospecific for aparticular hCBP epitope, represents a true measure of affinity.High-affinity antibody preparations with Ka ranging from about 10⁹ to10¹² l/mole are preferred for use in immunoassays in which thehCBP-antibody complex must withstand rigorous manipulations.Low-affinity antibody preparations with K_(a) ranging from about 10⁶ to10⁷ l/mole are preferred for use in immunopurification and similarprocedures which ultimately require dissociation of hCBP, preferably inactive form, from the antibody (Catty, D. (1988) Antibodies, Volume I: APractical Approach, IRL Press, Washington D.C.; Liddell, J. E. andCryer, A. (1991) A Practical Guide to Monoclonal Antibodies, John Wiley& Sons, New York N.Y.).

[0122] The titer and avidity of polyclonal antibody preparations may befurther evaluated to determine the quality and suitability of suchpreparations for certain downstream applications. For example, apolyclonal antibody preparation containing at least 1-2 mg specificantibody/ml, preferably 5-10 mg specific antibody/ml, is preferred foruse in procedures requiring precipitation of hCBP-antibody complexes.Procedures for evaluating antibody specificity, titer, and avidity, andguidelines for antibody quality and usage in various applications, aregenerally available. (See, e.g., Catty, supra, and Coligan et al.supra.)

[0123] In another embodiment of the invention, the polynucleotidesencoding hCBP, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, the complement of thepolynucleotide encoding hCBP may be used in situations in which it wouldbe desirable to block the transcription of the mRNA. In particular,cells may be transformed with sequences complementary to polynucleotidesencoding hCBP. Thus, complementary molecules or fragments may be used tomodulate hCBP activity, or to achieve regulation of gene function. Suchtechnology is now well known in the art, and sense or antisenseoligonucleotides or larger fragments can be designed from variouslocations along the coding or control regions of sequences encodinghCBP.

[0124] Expression vectors derived from retroviruses, adenoviruses, orherpes or vaccinia viruses, or from various bacterial plasmids, may beused for delivery of nucleotide sequences to the targeted organ, tissue,or cell population. Methods which are well known to those skilled in theart can be used to construct vectors to express nucleic acid sequencescomplementary to the polynucleotides encoding hCBP. (See, e.g.,Sambrook, supra; Ausubel, 1995, supra.)

[0125] Genes encoding hCBP can be turned off by transforming a cell ortissue with expression vectors which express high levels of apolynucleotide, or fragment thereof, encoding hCBP. Such constructs maybe used to introduce untranslatable sense or antisense sequences into acell. Even in the absence of integration into the DNA, such vectors maycontinue to transcribe RNA molecules until they are disabled byendogenous nucleases. Transient expression may last for a month or morewith a non-replicating vector, and may last even longer if appropriatereplication elements are part of the vector system.

[0126] As mentioned above, modifications of gene expression can beobtained by designing complementary sequences or antisense molecules(DNA, RNA, or PNA) to the control, 5′, or regulatory regions of the geneencoding hCBP. Oligonucleotides derived from the transcriptioninitiation site, e.g., between about positions −10 and +10 from thestart site, are preferred. Similarly, inhibition can be achieved usingtriple helix base-pairing methodology. Triple helix pairing is usefulbecause it causes inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature. (See, e.g., Gee, J. E. et al. (1994)in Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches,Futura Publishing, Mt. Kisco N.Y., pp. 163-177.) A complementarysequence or antisense molecule may also be designed to block translationof mRNA by preventing the transcript from binding to ribosomes.

[0127] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of sequences encodinghCBP.

[0128] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0129] Complementary ribonucleic acid molecules and ribozymes of theinvention may be prepared by any method known in the art for thesynthesis of nucleic acid molecules. These include techniques forchemically synthesizing oligonucleotides such as solid phasephosphoramidite chemical synthesis. Alternatively, RNA molecules may begenerated by in vitro and in vivo transcription of DNA sequencesencoding hCBP. Such DNA sequences may be incorporated into a widevariety of vectors with suitable RNA polymerase promoters such as T7 orSP6. Alternatively, these cDNA constructs that synthesize complementaryRNA, constitutively or inducibly, can be introduced into cell lines,cells, or tissues.

[0130] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule, or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0131] Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo, in vitro, and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection, by liposomeinjections, or by polycationic amino polymers may be achieved usingmethods which are well known in the art. (See, e.g., Goldman, C. K. etal. (1997) Nature Biotechnology 15:462-466.)

[0132] Any of the therapeutic methods described above may be applied toany subject in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0133] An additional embodiment of the invention relates to theadministration of a pharmaceutical or sterile composition, inconjunction with a pharmaceutically acceptable carrier, for any of thetherapeutic effects discussed above. Such pharmaceutical compositionsmay consist of hCBP, antibodies to hCBP, and mimetics, agonists,antagonists, or inhibitors of hCBP. The compositions may be administeredalone or in combination with at least one other agent, such as astabilizing compound, which may be administered in any sterile,biocompatible pharmaceutical carrier including, but not limited to,saline, buffered saline, dextrose, and water. The compositions may beadministered to a patient alone, or in combination with other agents,drugs, or hormones.

[0134] The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

[0135] In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing, Easton Pa.).

[0136] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

[0137] Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Suitable auxiliaries can be added, if desired.Suitable excipients include carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, and sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

[0138] Dragee cores may be used in conjunction with suitable coatings,such as concentrated sugar solutions, which may also contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

[0139] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with fillers or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0140] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils, such as sesame oil, orsynthetic fatty acid esters, such as ethyl oleate, triglycerides, orliposomes. Non-lipid polycationic amino polymers may also be used fordelivery. Optionally, the suspension may also contain suitablestabilizers or agents to increase the solubility of the compounds andallow for the preparation of highly concentrated solutions.

[0141] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0142] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0143] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tendto be more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7%mannitol, at a pH range of 4.5 to 5.5, that is combined with bufferprior to use.

[0144] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of hCBP, such labeling wouldinclude amount, frequency, and method of administration.

[0145] Pharmaceutical compositions suitable for use in the inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0146] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells or in animal models such as mice, rats, rabbits, dogs, or pigs. Ananimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0147] A therapeutically effective dose refers to that amount of activeingredient, for example hCBP or fragments thereof, antibodies of hCBP,and agonists, antagonists or inhibitors of hCBP, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by calculating the ED₅₀ (the dosetherapeutically effective in 50% of the population) or LD₅₀ (the doselethal to 50% of the population) statistics. The dose ratio of toxic totherapeutic effects is the therapeutic index, and it can be expressed asthe LD₅₀/ED₅₀ ratio. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred. The data obtained from cell cultureassays and animal studies are used to formulate a range of dosage forhuman use. The dosage contained in such compositions is preferablywithin a range of circulating concentrations that includes the ED₅₀ withlittle or no toxicity. The dosage varies within this range dependingupon the dosage form employed, the sensitivity of the patient, and theroute of administration.

[0148] The exact dosage will be determined by the practitioner, in lightof factors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Long-acting pharmaceuticalcompositions may be administered every 3 to 4 days, every week, orbiweekly depending on the half-life and clearance rate of the particularformulation.

[0149] Normal dosage amounts may vary from about 0.1 μg to 100,000 μg,up to a total dose of about 1 gram, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0150] Diagnostics

[0151] In another embodiment, antibodies which specifically bind hCBPmay be used for the diagnosis of disorders characterized by expressionof hCBP, or in assays to monitor patients being treated with hCBP oragonists, antagonists, or inhibitors of hCBP. Antibodies useful fordiagnostic purposes may be prepared in the same manner as describedabove for therapeutics. Diagnostic assays for hCBP include methods whichutilize the antibody and a label to detect hCBP in human body fluids orin extracts of cells or tissues. The antibodies may be used with orwithout modification, and may be labeled by covalent or non-covalentattachment of a reporter molecule. A wide variety of reporter molecules,several of which are described above, are known in the art and may beused.

[0152] A variety of protocols for measuring hCBP, including ELISAs,RIAs, and FACS, are known in the art and provide a basis for diagnosingaltered or abnormal levels of hCBP expression. Normal or standard valuesfor hCBP expression are established by combining body fluids or cellextracts taken from normal mammalian subjects, preferably human, withantibody to hCBP under conditions suitable for complex formation. Theamount of standard complex formation may be quantitated by variousmethods, preferably by photometric means. Quantities of hCBP expressedin subject, control, and disease samples from biopsied tissues arecompared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

[0153] In another embodiment of the invention, the polynucleotidesencoding hCBP may be used for diagnostic purposes. The polynucleotideswhich may be used include oligonucleotide sequences, complementary RNAand DNA molecules, and PNAs. The polynucleotides may be used to detectand quantitate gene expression in biopsied tissues in which expressionof hCBP may be correlated with disease. The diagnostic assay may be usedto determine absence, presence, and excess expression of hCBP, and tomonitor regulation of hCBP levels during therapeutic intervention.

[0154] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding hCBP or closely related molecules may be used to identifynucleic acid sequences which encode hCBP. The specificity of the probe,whether it is made from a highly specific region, e.g., the 5′regulatory region, or from a less specific region, e.g., a conservedmotif, and the stringency of the hybridization or amplification(maximal, high, intermediate, or low), will determine whether the probeidentifies only naturally occurring sequences encoding hCBP, allelicvariants, or related sequences.

[0155] Probes may also be used for the detection of related sequences,and should preferably have at least 50% sequence identity to any of thehCBP encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and may be derived from the sequence of SEQID NO:2 or from genomic sequences including promoters, enhancers, andintrons of the hCBP gene.

[0156] Means for producing specific hybridization probes for DNAsencoding hCBP include the cloning of polynucleotide sequences encodinghCBP or hCBP derivatives into vectors for the production of mRNA probes.Such vectors are known in the art, are commercially available, and maybe used to synthesize RNA probes in vitro by means of the addition ofthe appropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, by radionuclides such as ³²P or ³⁵S, or by enzymatic labels,such as alkaline phosphatase coupled to the probe via avidin/biotincoupling systems, and the like.

[0157] Polynucleotide sequences encoding hCBP may be used for thediagnosis of disorders associated with expression of hCBP. Examples ofsuch disorders include, but are not limited to, a cancer, such asadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; a reproductive disordersuch as disorders of prolactin production, infertility, including tubaldisease, ovulatory defects, and endometriosis, disruptions of theestrous cycle, disruptions of the menstrual cycle, polycystic ovarysyndrome, ovarian hyperstimulation syndrome, endometrial and ovariantumors, uterine fibroids, autoimmune disorders, ectopic pregnancies, andteratogenesis, cancer of the breast, fibrocystic breast disease, andgalactorrhea, disruptions of spermatogenesis, abnormal sperm physiology,cancer of the testis, cancer of the prostate, benign prostatichyperplasia, prostatitis, Peyronie's disease, impotence, carcinoma ofthe male breast, and gynecomastia; a developmental disorder, such asrenal tubular acidosis, anemia, Cushing's syndrome, achondroplasticdwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadaldysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinaryabnormalities, and mental retardation), Smith-Magenis syndrome,myelodysplastic syndrome, hereditary mucoepithelial dysplasia,hereditary keratodermas, hereditary neuropathies such asCharcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism,hydrocephalus, seizure disorders such as Syndenham's chorea and cerebralpalsy, spina bifida, anencephaly, craniorachischisis, congenitalglaucoma, cataract, and sensorineural hearing loss; and an immunedisorder, such as acquired immunodeficiency syndrome (AIDS), Addison'sdisease, adult respiratory distress syndrome, allergies, ankylosingspondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmunehemolytic anemia, autoimmune thyroiditis, autoimmunepolyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED),bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopicdermatitis, dermatomyositis, diabetes mellitus, emphysema, episodiclymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythemanodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome,gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,irritable bowel syndrome, multiple sclerosis, myasthenia gravis,myocardial or pericardial inflammation, osteoarthritis, osteoporosis,pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoidarthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis,systemic lupus erythematosus, systemic sclerosis, thrombocytopenicpurpura, ulcerative colitis, uveitis, Werner syndrome, complications ofcancer, hemodialysis, and extracorporeal circulation, viral, bacterial,fungal, parasitic, protozoal, and helminthic infections, and trauma. Thepolynucleotide sequences encoding hCBP may be used in Southern ornorthern analysis, dot blot, or other membrane-based technologies; inPCR technologies; in dipstick, pin, and multiformat ELISA-like assays;and in microarrays utilizing fluids or tissues from patients to detectaltered hCBP expression. Such qualitative or quantitative methods arewell known in the art.

[0158] In a particular aspect, the nucleotide sequences encoding hCBPmay be useful in assays that detect the presence of associateddisorders, particularly those mentioned above. The nucleotide sequencesencoding hCBP may be labeled by standard methods and added to a fluid ortissue sample from a patient under conditions suitable for the formationof hybridization complexes. After a suitable incubation period, thesample is washed and the signal is quantitated and compared with astandard value. If the amount of signal in the patient sample issignificantly altered in comparison to a control sample then thepresence of altered levels of nucleotide sequences encoding hCBP in thesample indicates the presence of the associated disorder. Such assaysmay also be used to evaluate the efficacy of a particular therapeutictreatment regimen in animal studies, in clinical trials, or to monitorthe treatment of an individual patient.

[0159] In order to provide a basis for the diagnosis of a disorderassociated with expression of hCBP, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, encoding hCBP, underconditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with values from an experiment in which a known amountof a substantially purified polynucleotide is used. Standard valuesobtained in this manner may be compared with values obtained fromsamples from patients who are symptomatic for a disorder. Deviation fromstandard values is used to establish the presence of a disorder.

[0160] Once the presence of a disorder is established and a treatmentprotocol is initiated, hybridization assays may be repeated on a regularbasis to determine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0161] With respect to cancer, the presence of an abnormal amount oftranscript (either under- or over-expressed) in biopsied tissue from anindividual may indicate a predisposition for the development of thedisease, or may provide a means for detecting the disease prior to theappearance of actual clinical symptoms. A more definitive diagnosis ofthis type may allow health professionals to employ preventative measuresor aggressive treatment earlier thereby preventing the development orfurther progression of the cancer.

[0162] Additional diagnostic uses for oligonucleotides designed from thesequences encoding hCBP may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding hCBP, or a fragment of a polynucleotide complementary to thepolynucleotide encoding hCBP, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantitation of closely related DNA or RNA sequences.

[0163] Methods which may also be used to quantitate the expression ofhCBP include radiolabeling or biotinylating nucleotides, coamplificationof a control nucleic acid, and interpolating results from standardcurves. (See, e.g., Melby, P. C. et al. (1993) J. Immunol. Methods159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 212:229-236.) Thespeed of quantitation of multiple samples may be accelerated by runningthe assay in an ELISA format where the oligomer of interest is presentedin various dilutions and a spectrophotometric or calorimetric responsegives rapid quantitation.

[0164] In further embodiments, oligonucleotides or longer fragmentsderived from any of the polynucleotide sequences described herein may beused as targets in a microarray. The microarray can be used to monitorthe expression level of large numbers of genes simultaneously and toidentify genetic variants, mutations, and polymorphisms. Thisinformation may be used to determine gene function, to understand thegenetic basis of a disorder, to diagnose a disorder, and to develop andmonitor the activities of therapeutic agents.

[0165] Microarrays may be prepared, used, and analyzed using methodsknown in the art. (See, e.g., Brennan, T. M. et al. (1995) U.S. Pat. No.5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci.93:10614-10619; Baldeschweiler et al. (1995) PCT applicationWO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505;Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155; andHeller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.)

[0166] In another embodiment of the invention, nucleic acid sequencesencoding hCBP may be used to generate hybridization probes useful inmapping the naturally occurring genomic sequence. The sequences may bemapped to a particular chromosome, to a specific region of a chromosome,or to artificial chromosome constructions, e.g., human artificialchromosomes (HACs), yeast artificial chromosomes (YACs), bacterialartificial chromosomes (BACs), bacterial P1 constructions, or singlechromosome cDNA libraries. (See, e.g., Harrington, J. J. et al. (1997)Nat Genet. 15:345-355; Price, C. M. (1993) Blood Rev. 7:127-134; andTrask, B.J. (1991) Trends Genet. 7:149-154.)

[0167] Fluorescent in situ hybridization (FISH) may be correlated withother physical chromosome mapping techniques and genetic map data. (See,e.g., Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968.)Examples of genetic map data can be found in various scientific journalsor at the Online Mendelian Inheritance in Man (OMIM) site. Correlationbetween the location of the gene encoding hCBP on a physical chromosomalmap and a specific disorder, or a predisposition to a specific disorder,may help define the region of DNA associated with that disorder. Thenucleotide sequences of the invention may be used to detect differencesin gene sequences among normal, carrier, and affected individuals.

[0168] In situ hybridization of chromosomal preparations and physicalmapping techniques, such as linkage analysis using establishedchromosomal markers, may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms by physical mapping. This provides valuableinformation to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, e.g., ataxia-telangiectasia to 11q22-23, anysequences mapping to that area may represent associated or regulatorygenes for further investigation. (See, e.g., Gatti, R. A. et al. (1988)Nature 336:577-580.) The nucleotide sequence of the subject inventionmay also be used to detect differences in the chromosomal location dueto translocation, inversion, etc., among normal, carrier, or affectedindividuals.

[0169] In another embodiment of the invention, hCBP, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes between hCBPand the agent being tested may be measured.

[0170] Another technique for drug screening provides for high throughputscreening of compounds having suitable binding affinity to the proteinof interest. (See, e.g., Geysen, et al. (1984) PCT applicationWO84/03564.) In this method, large numbers of different small testcompounds are synthesized on a solid substrate. The test compounds arereacted with hCBP, or fragments thereof, and washed. Bound hCBP is thendetected by methods well known in the art. Purified hCBP can also becoated directly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support.

[0171] In another embodiment, one may use competitive drug screeningassays in which neutralizing antibodies capable of binding hCBPspecifically compete with a test compound for binding hCBP. In thismanner, antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with hCBP.

[0172] In additional embodiments, the nucleotide sequences which encodehCBP may be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

[0173] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention.

EXAMPLES

[0174] I. SMCCNOS01 cDNA Library Construction

[0175] The SMCCNOS01 subtracted coronary artery smooth muscle celllibrary was constructed using 7.56×10⁶ clones from the SMCCNOT02 libraryand was subjected to two rounds of subtraction hybridization for 48hours with 6.12×10⁶ clones from SMCCNOT01. The starting library forsubtraction was constructed using RNA isolated from coronary arterysmooth muscle cells removed from a 3-year-old Caucasian male. The cellswere treated with TNFα and IL-1β 10 ng/ml each for 20 hours. Thehybridization probe for subtraction was derived from a similarlyconstructed library from RNA isolated from untreated coronary arterysmooth muscle cells from the same donor. Subtractive hybridizationconditions were based on the methodologies of Swaroop et al. NAR (1991)19:1954, and Bonaldo et al. Genome Research (1996) 6:791.

[0176] For both cDNA libraries SMCCNOT01 and SMCCNOT02, the frozencoronary artery smooth muscle cells (50-100 mg) were homogenized in GTChomogenization buffer (4.0M guanidine thiocyanate, 0.1M Tris-HCl pH 7.5,1% 2-Mercaptoethanol). Two volumes of binding buffer (0.4M LiCl, 0.1MTris-HCI pH 7.5, 0.02M EDTA) were added and the resulting mixturevortexed at 13,000 rpm. The supernatant was removed and combined withOligo(dT)₂₅ bound MPG Streptavidin particles. After rotation at roomtemperature, the mRNA-Oligo(dT)₂₅-Streptavidin particles were separatedfrom the supernatant, washed twice with hybridization buffer I (0.15MNaCl, 0.01M Tris-HCl pH8.0, 1 mM EDTA, 0.1% Lauryl Sarcosinate) andwashed twice with hybridization buffer II (0.15M NaCl, 0.01M Tris-HCIpH8.0, 1 mM EDTA) using magnetic separation at each step to remove thesupernatant from the particles. Bound mRNA was eluted from the MPGStreptavidin particles with release solution and heated to 65° C. Thesupernatant containing eluted mRNA was magnetically separated from theStreptavidin particles and used to construct the cDNA libraries.

[0177] Poly(A+) RNA was used for cDNA synthesis and construction of thecDNA library according to the recommended protocols in the SUPERSCRIPTplasmid system (Life Technologies). The cDNAs were fractionated on aSEPHAROSE CL4B column (Amersham Pharmacia Biotech), and those cDNAsexceeding 400 bp were ligated into pINCY (Incyte Pharmaceuticals, PaloAlto, Calif.). Recombinant plasmids were transformed into DH5α competentcells or ELECTROMAX cells (Life Technologies).

[0178] II. Isolation of cDNA Clones

[0179] Plasmid DNA was released from the cells and purified using theR.E.A.L. PREP 96 plasmid kit (QIAGEN). The recommended protocol wasemployed except for the following changes: 1) the bacteria were culturedin 1 ml of sterile Terrific Broth (Life Technologies) with carbenicillinat 25 mg/l and glycerol at 0.4%; 2) after the cultures were incubatedfor 19 hours, the cells were lysed with 0.3 ml of lysis buffer; and 3)following isopropanol precipitation, the plasmid DNA pellets were eachresuspended in 0.1 ml of distilled water. The DNA samples were stored at4° C.

[0180] III. Sequencing and Analysis

[0181] The cDNAs were prepared for sequencing using the ABI CATALYST 800(Perkin-Elmer) or the HYDRA microdispenser (Robbins Scientific) orMICROLAB 2200 (Hamilton) systems in combination with the PTC-200 thermalcyclers (MJ Research). The cDNAs were sequenced using the ABI PRISM 373or 377 sequencing systems (Perkin-Elmer) and standard ABI protocols,base calling software, and kits. In one alternative, cDNAs weresequenced using the MEGABACE 1000 DNA sequencing system (MolecularDynamics). In another alternative, the cDNAs were amplified andsequenced using the ABI PRISM BIGDYE Terminator cycle sequencing readyreaction kit (Perkin-Elmer). In yet another alternative, cDNAs weresequenced using solutions and dyes from Amersham Pharmacia Biotech.Reading frames for the ESTs were determined using standard methods(reviewed in Ausubel, 1997, supra, unit 7.7). Some of the cDNA sequenceswere selected for extension using the techniques disclosed in Example V.

[0182] The polynucleotide sequences derived from cDNA, extension, andshotgun sequencing were assembled and analyzed using a combination ofsoftware programs which utilize algorithms well known to those skilledin the art. Table 1 summarizes the software programs, descriptions,references, and threshold parameters used. The first column of Table 1shows the tools, programs, and algorithms used, the second columnprovides a brief description thereof, the third column presents thereferences which are incorporated by reference herein, and the fourthcolumn presents, where applicable, the scores, probability values, andother parameters used to evaluate the strength of a match between twosequences (the higher the probability the greater the homology).Sequences were analyzed using MACDNASIS PRO software (Hitachi SoftwareEngineering) and LASERGENE software (DNASTAR).

[0183] The polynucleotide sequences were validated by removing vector,linker, and polyA sequences and by masking ambiguous bases, usingalgorithms and programs based on BLAST, dynamic programming, anddinucleotide nearest neighbor analysis. The sequences were then queriedagainst a selection of public databases such as GenBank primate, rodent,mammalian, vertebrate, and eukaryote databases, and BLOCKS to acquireannotation, using programs based on BLAST, FASTA, and BLIMPS. Thesequences were assembled into full length polynucleotide sequences usingprograms based on Phred, Phrap, and Consed, and were screened for openreading frames using programs based on GeneMark, BLAST, and FASTA. Thefull length polynucleotide sequences were translated to derive thecorresponding full length amino acid sequences, and these full lengthsequences were subsequently analyzed by querying against databases suchas the GenBank databases (described above), SwissProt, BLOCKS, PRINTS,Prosite, and Hidden Markov Model (HMM)-based protein family databasessuch as PFAM. HMM is a probabilistic approach which analyzes consensusprimary structures of gene families. (See, e.g., Eddy, S. R. (1996) Cur.Opin. Str. Biol. 6:361-365.)

[0184] The programs described above for the assembly and analysis offull length polynucleotide and amino acid sequences were used toidentify polynucleotide sequence fragments from SEQ ID NO:2. Fragmentsfrom about 20 to about 4000 nucleotides which are useful inhybridization and amplification technologies were described in TheInvention section above.

[0185] IV. Northern Analysis

[0186] Northern analysis is a laboratory technique used to detect thepresence of a transcript of a gene and involves the hybridization of alabeled nucleotide sequence to a membrane on which RNAs from aparticular cell type or tissue have been bound. (See, e.g., Sambrook,supra, ch. 7; Ausubel, 1995, supra, ch. 4 and 16.)

[0187] Analogous computer techniques applying BLAST were used to searchfor identical or related molecules in nucleotide databases such asGenBank or LIFESEQ database (Incyte Pharmaceuticals, Palo Alto Calif.).This analysis is much faster than multiple membrane-basedhybridizations. In addition, the sensitivity of the computer search canbe modified to determine whether any particular match is categorized asexact or similar. The basis of the search is the product score, which isdefined as:$\frac{\text{\%~~sequence~~identity} \times \text{\%~~maximum~~BLAST~~score}}{100}$

[0188] The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1% to 2% error, and, with a product score of 70, the match will beexact. Similar molecules are usually identified by selecting those whichshow product scores between 15 and 40, although lower scores mayidentify related molecules.

[0189] The results of northern analyses are reported as a percentagedistribution of libraries in which the transcript encoding hCBPoccurred. Analysis involved the categorization of cDNA libraries byorgan/tissue and disease. The organ/tissue categories includedcardiovascular, dermatologic, developmental, endocrine,gastrointestinal, hematopoietic/immune, musculoskeletal, nervous,reproductive, and urologic. The disease/condition categories includedcancer, inflammation/trauma, cell proliferation, neurological, andpooled. For each category, the number of libraries expressing thesequence of interest was counted and divided by the total number oflibraries across all categories. Percentage values of tissue-specificand disease- or condition-specific expression are reported in thedescription of the invention.

[0190] V. Extension of hCBP Encoding Polynucleotides

[0191] The full length nucleic acid sequence of SEQ ID NO:2 was producedby extension of an appropriate fragment of the full length moleculeusing oligonucleotide primers designed from this fragment. One primerwas synthesized to initiate 5′ extension of the known fragment, and theother primer, to initiate 3′ extension of the known fragment. Theinitial primers were designed using OLIGO 4.06 software (NationalBiosciences), or another appropriate program, to be about 22 to 30nucleotides in length, to have a GC content of about 50% or more, and toanneal to the target sequence at temperatures of about 68° C. to about72° C. Any stretch of nucleotides which would result in hairpinstructures and primer-primer dimerizations was avoided.

[0192] Selected human cDNA libraries were used to extend the sequence.If more than one extension was necessary or desired, additional ornested sets of primers were designed.

[0193] High fidelity amplification was obtained by PCR using methodswell known in the art. PCR was performed in 96-well plates using thePTC-200 thermal cycler (MJ Research, Inc.). The reaction mix containedDNA template, 200 nmol of each primer, reaction buffer containing Mg²⁺,(NH₄)₂SO₄, and β-mercaptoethanol, Taq DNA polymerase (Amersham PharmaciaBiotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase(Stratagene), with the following parameters for primer pair PCI A andPCI B: Step 1: 94° C., 3 min; Step 2: 94° C., 15 sec; Step 3: 60° C., 1min; Step 4: 68° C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times;Step 6: 68 ° C., 5 min; Step 7: storage at 4 ° C. In the alternative,the parameters for primer pair T7 and SK+were as follows: Step 1: 94 °C., 3 min; Step 2: 94° C. 15 sec; Step 3: 57° C., 1 min; Step 4: 68° C.,2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68 ° C., 5min; Step 7: storage at 4° C.

[0194] The concentration of DNA in each well was determined bydispensing 100 μl PICO GREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene Oreg.) dissolved in 1× TE and 0.5 μl ofundiluted PCR product into each well of an opaque fluorimeter plate(Corning Costar, Acton Mass.), allowing the DNA to bind to the reagent.The plate was scanned in a Fluoroskan II (Labsystems Oy, Helsinki,Finland) to measure the fluorescence of the sample and to quantify theconcentration of DNA. A 5 μl to 10 μl aliquot of the reaction mixturewas analyzed by electrophoresis on a 1% agarose mini-gel to determinewhich reactions were successful in extending the sequence.

[0195] The extended nucleotides were desalted and concentrated,transferred to 384-well plates, digested with CviJI cholera virusendonuclease (Molecular Biology Research, Madison Wis.), and sonicatedor sheared prior to religation into pUC 18 vector (Amersham PharmaciaBiotech). For shotgun sequencing, the digested nucleotides wereseparated on low concentration (0.6 to 0.8%) agarose gels, fragmentswere excised, and agar digested with Agar ACE (Promega). Extended cloneswere religated using T4 ligase (New England Biolabs, Beverly Mass.) intopUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNApolymerase (Stratagene) to fill-in restriction site overhangs, andtransfected into competent E. coli cells. Transformed cells wereselected on antibiotic-containing media, individual colonies were pickedand cultured overnight at 37 ° C. in 384-well plates in LB/2× carbliquid media.

[0196] The cells were lysed, and DNA was amplified by PCR using Taq DNApolymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase(Stratagene) with the following parameters: Step 1: 94 ° C., 3 min; Step2: 94° C., 15 sec; Step 3: 60° C., 1 min; Step 4: 72° C., 2 min; Step 5:steps 2, 3, and 4 repeated 29 times; Step 6: 72° C., 5 min; Step 7:storage at 4° C. DNA was quantified by PICOGREEN reagent (MolecularProbes) as described above. Samples with low DNA recoveries werereamplified using the same conditions as described above. Samples werediluted with 20% dimethysulphoxide (1:2, v/v), and sequenced usingDYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit(Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator cyclesequencing ready reaction kit (Perkin-Elmer).

[0197] In like manner, the nucleotide sequence of SEQ ID NO:2 is used toobtain 5′ regulatory sequences using the procedure above,oligonucleotides designed for such extension, and an appropriate genomiclibrary.

[0198] VI. Labeling and use of Individual Hybridization Probes

[0199] Hybridization probes derived from SEQ ID NO:2 are employed toscreen cDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 software (National Biosciences) and labeled bycombining 50 μmol of each oligomer, 250 μCi of [³²P]-adenosinetriphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase(DuPont NEN, Boston Mass.). The labeled oligonucleotides aresubstantially purified using a SEPHADEX G-25 superfine size exclusiondextran bead column (Amersham Pharmacia Biotech). An aliquot containing10⁷ counts per minute of the labeled probe is used in a typicalmembrane-based hybridization analysis of human genomic DNA digested withone of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba I,or Pvu II (DuPont NEN).

[0200] The DNA from each digest is fractionated on a 0.7% agarose geland transferred to nylon membranes (NYTRAN PLUS, Schleicher & Schuell,Durham NH). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1× salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT-AR film(Eastman Kodak, Rochester N.Y.) is exposed to the blots, hybridizationpatterns are compared visually.

[0201] VII. Microarrays

[0202] A chemical coupling procedure and an ink jet device can be usedto synthesize array elements on the surface of a substrate. (See, e.g.,Baldeschweiler, supra.) An array analogous to a dot or slot blot mayalso be used to arrange and link elements to the surface of a substrateusing thermal, UV, chemical, or mechanical bonding procedures. A typicalarray may be produced by hand or using available methods and machinesand contain any appropriate number of elements. After hybridization,nonhybridized probes are removed and a scanner used to determine thelevels and patterns of fluorescence. The degree of complementarity andthe relative abundance of each probe which hybridizes to an element onthe microarray may be assessed through analysis of the scanned images.

[0203] Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragmentsthereof may comprise the elements of the microarray. Fragments suitablefor hybridization can be selected using software well known in the artsuch as LASERGENE software (DNASTAR). Full-length cDNAs, ESTs, orfragments thereof corresponding to one of the nucleotide sequences ofthe present invention, or selected at random from a cDNA libraryrelevant to the present invention, are arranged on an appropriatesubstrate, e.g., a glass slide. The cDNA is fixed to the slide using,e.g., UV cross-linking followed by thermal and chemical treatments andsubsequent drying. (See, e.g., Schena, M. et al. (1995) Science270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645.)Fluorescent probes are prepared and used for hybridization to theelements on the substrate. The substrate is analyzed by proceduresdescribed above.

[0204] VIII. Complementary Polynucleotides

[0205] Sequences complementary to the hCBP-encoding sequences, or anyparts thereof, are used to detect, decrease, or inhibit expression ofnaturally occurring hCBP. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO 4.06 software(National Biosciences) and the coding sequence of hCBP. To inhibittranscription, a complementary oligonucleotide is designed from the mostunique 5′ sequence and used to prevent promoter binding to the codingsequence. To inhibit translation, a complementary oligonucleotide isdesigned to prevent ribosomal binding to the hCBP-encoding transcript.

[0206] IX. Expression of hCBP

[0207] Expression and purification of hCBP are achieved using bacterialor virus-based expression systems. For expression of hCBP in bacteria,cDNA is subcloned into an appropriate vector containing an antibioticresistance gene and an inducible promoter that directs high levels ofcDNA transcription. Examples of such promoters include, but are notlimited to, the trp-lac (tac) hybrid promoter and the T5 or T7bacteriophage promoter in conjunction with the lac operator regulatoryelement. Recombinant vectors are transformed into suitable bacterialhosts, e.g., BL21(DE3). Antibiotic resistant bacteria express hCBP uponinduction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expressionof hCBP in eukaryotic cells is achieved by infecting insect or mammaliancell lines with recombinant Autographica californica nuclearpolyhedrosis virus (AcMNPV), commonly known as baculovirus. Thenonessential polyhedrin gene of baculovirus is replaced with cDNAencoding hCBP by either homologous recombination or bacterial-mediatedtransposition involving transfer plasmid intermediates. Viralinfectivity is maintained and the strong polyhedrin promoter drives highlevels of cDNA transcription. Recombinant baculovirus is used to infectSpodoptera frugiperda (Sf9) insect cells in most cases, or humanhepatocytes, in some cases. Infection of the latter requires additionalgenetic modifications to baculovirus. (See Engelhard, E. K. et al.(1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)Hum. Gene Ther. 7:1937-1945.)

[0208] In most expression systems, hCBP is synthesized as a fusionprotein with, e.g., glutathione S-transferase (GST) or a peptide epitopetag, such as FLAG or 6-His, permitting rapid, single-step,affinity-based purification of recombinant fusion protein from crudecell lysates. GST, a 26-kilodalton enzyme from Schistosoma japonicum,enables the purification of fusion proteins on immobilized glutathioneunder conditions that maintain protein activity and antigenicity(Amersham Pharmacia Biotech). Following purification, the GST moiety canbe proteolytically cleaved from hCBP at specifically engineered sites.FLAG, an 8-amino acid peptide, enables immunoaffinity purification usingcommercially available monoclonal and polyclonal anti-FLAG antibodies(Eastman Kodak). 6-His, a stretch of six consecutive histidine residues,enables purification on metal-chelate resins (QIAGEN). Methods forprotein expression and purification are discussed in Ausubel (1995,supra, ch 10 and 16). Purified hCBP obtained by these methods can beused directly in the following activity assay.

[0209] X. Demonstration of hCBP Activity

[0210] The assay for human calcium-binding proteins is based upon theability of hCBPs to down-regulate mitosis. hCBP can be expressed bytransforming a mammalian cell line such as COS7, HeLa or CHO with aneukaryotic expression vector encoding hCBP. Eukaryotic expressionvectors are commercially available, and the techniques to introduce theminto cells are well known to those skilled in the art. The cells areincubated for 48-72 hours after transformation under conditionsappropriate for the cell line to allow expression of hCBP. Then, phasemicroscopy is used to compare the mitotic index of transformed versuscontrol cells. The decrease in the mitotic index is proportional to thehCBP activity.

[0211] XI. Functional Assays

[0212] hCBP function is assessed by expressing the sequences encodinghCBP at physiologically elevated levels in mammalian cell culturesystems. cDNA is subcloned into a mammalian expression vector containinga strong promoter that drives high levels of cDNA expression. Vectors ofchoice include pCMV SPORT (Life Technologies) and pCR3.1 (Invitrogen,Carlsbad Calif.), both of which contain the cytomegalovirus promoter.5-10 μg of recombinant vector are transiently transfected into a humancell line, preferably of endothelial or hematopoietic origin, usingeither liposome formulations or electroporation. 1-2 μg of an additionalplasmid containing sequences encoding a marker protein areco-transfected. Expression of a marker protein provides a means todistinguish transfected cells from nontransfected cells and is areliable predictor of cDNA expression from the recombinant vector.Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP;Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), anautomated, laser optics-based technique, is used to identify transfectedcells expressing GFP or CD64-GFP, and to evaluate cellular properties,for example, their apoptotic state. FCM detects and quantifies theuptake of fluorescent molecules that diagnose events preceding orcoincident with cell death. These events include changes in nuclear DNAcontent as measured by staining of DNA with propidium iodide; changes incell size and granularity as measured by forward light scatter and 90degree side light scatter; down-regulation of DNA synthesis as measuredby decrease in bromodeoxyuridine uptake; alterations in expression ofcell surface and intracellular proteins as measured by reactivity withspecific antibodies; and alterations in plasma membrane composition asmeasured by the binding of fluorescein-conjugated Annexin V protein tothe cell surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994) Flow Cytometry, Oxford, New York N.Y.

[0213] The influence of hCBP on gene expression can be assessed usinghighly purified populations of cells transfected with sequences encodinghCBP and either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on thesurface of transfected cells and bind to conserved regions of humanimmunoglobulin G (IgG). Transfected cells are efficiently separated fromnontransfected cells using magnetic beads coated with either human IgGor antibody against CD64 (DYNAL, Lake Success NY). mRNA can be purifiedfrom the cells using methods well known by those of skill in the art.Expression of MRNA encoding hCBP and other genes of interest can beanalyzed by northern analysis or microarray techniques.

[0214] XII. Production of hCBP Specific Antibodies

[0215] hCBP substantially purified using polyacrylamide gelelectrophoresis (PAGE; see, e.g., Harrington, M. G. (1990) MethodsEnzymol. 182:488-495), or other purification techniques, is used toimmunize rabbits and to produce antibodies using standard protocols.

[0216] Alternatively, the hCBP amino acid sequence is analyzed usingLASERGENE software (DNASTAR) to determine regions of highimmunogenicity, and a corresponding oligopeptide is synthesized and usedto raise antibodies by means known to those of skill in the art. Methodsfor selection of appropriate epitopes, such as those near the C-terminusor in hydrophilic regions are well described in the art. (See, e.g.,Ausubel, 1995, supra, ch. 11.)

[0217] Typically, oligopeptides 15 residues in length are synthesizedusing an ABI 431A peptide synthesizer (Perkin-Elmer) usingfmoc-chemistry and coupled to KLH (Sigma-Aldrich, St. Louis Mo.) byreaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) toincrease immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits areimmunized with the oligopeptide-KLH complex in complete Freund'sadjuvant. Resulting antisera are tested for antipeptide activity by, forexample, binding the peptide to plastic, blocking with 1% BSA, reactingwith rabbit antisera, washing, and reacting with radio-iodinated goatanti-rabbit IgG.

[0218] XIII. Purification of Naturally Occurring hCBP Using SpecificAntibodies

[0219] Naturally occurring or recombinant hCBP is substantially purifiedby immunoaffinity chromatography using antibodies specific for hCBP. Animmunoaffinity column is constructed by covalently coupling anti-hCBPantibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin isblocked and washed according to the manufacturer's instructions.

[0220] Media containing hCBP are passed over the immunoaffinity column,and the column is washed under conditions that allow the preferentialabsorbance of hCBP (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/hCBP binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), and hCBPis collected.

[0221] XIV. Identification of Molecules Which Interact with hCBP

[0222] hCBP, or biologically active fragments thereof, are labeled with1251 Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J.133:529-539.) Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled hCBP, washed, and anywells with labeled hCBP complex are assayed. Data obtained usingdifferent concentrations of hCBP are used to calculate values for thenumber, affinity, and association of hCBP with the candidate molecules.Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

1 5 1 337 PRT Homo sapiens 3734805 1 Met Lys Lys Met Pro Leu Phe Ser LysSer His Lys Asn Pro Ala 1 5 10 15 Glu Ile Val Lys Ile Leu Lys Asp AsnLeu Ala Ile Leu Glu Lys 20 25 30 Gln Asp Lys Lys Thr Asp Lys Ala Ser GluGlu Val Ser Lys Ser 35 40 45 Leu Gln Ala Met Lys Glu Ile Leu Cys Gly ThrAsn Glu Lys Glu 50 55 60 Pro Pro Thr Glu Ala Val Ala Gln Leu Ala Gln GluLeu Tyr Ser 65 70 75 Ser Gly Leu Leu Val Thr Leu Ile Ala Asp Leu Gln LeuIle Asp 80 85 90 Phe Glu Gly Lys Lys Asp Val Thr Gln Ile Phe Asn Asn IleLeu 95 100 105 Arg Arg Gln Ile Gly Thr Arg Ser Pro Thr Val Glu Tyr IleSer 110 115 120 Ala His Pro His Ile Leu Phe Met Leu Leu Lys Gly Tyr GluAla 125 130 135 Pro Gln Ile Ala Leu Arg Cys Gly Ile Met Leu Arg Glu CysIle 140 145 150 Arg His Glu Pro Leu Ala Lys Ile Ile Leu Phe Ser Asn GlnPhe 155 160 165 Arg Asp Phe Phe Lys Tyr Val Glu Leu Ser Thr Phe Asp IleAla 170 175 180 Ser Asp Ala Phe Ala Thr Phe Lys Asp Leu Leu Thr Arg HisLys 185 190 195 Val Leu Val Ala Asp Phe Leu Glu Gln Asn Tyr Asp Thr IlePhe 200 205 210 Glu Asp Tyr Glu Lys Leu Leu Gln Ser Glu Asn Tyr Val ThrLys 215 220 225 Arg Gln Ser Leu Lys Leu Leu Gly Glu Leu Ile Leu Asp ArgHis 230 235 240 Asn Phe Ala Ile Met Thr Lys Tyr Ile Ser Lys Pro Glu AsnLeu 245 250 255 Lys Leu Met Met Asn Leu Leu Arg Asp Lys Ser Pro Asn IleGln 260 265 270 Phe Glu Ala Phe His Val Phe Lys Val Phe Val Ala Ser ProHis 275 280 285 Lys Thr Gln Pro Ile Val Glu Ile Leu Leu Lys Asn Gln ProLys 290 295 300 Leu Ile Glu Phe Leu Ser Ser Phe Gln Lys Glu Arg Thr AspAsp 305 310 315 Glu Gln Phe Ala Asp Glu Lys Asn Tyr Leu Ile Lys Gln IleArg 320 325 330 Asp Leu Lys Lys Thr Ala Pro 335 2 1344 DNA Homo sapiens3734805 2 cagaaacaga actgcctgtg acagattaag agacaagcaa ggcttggaatctgagagcaa 60 gcaaagagag tggaaattta cagctgcctt atcattccat attggaagaagagatttcta 120 cacatgaaaa aaatgccttt gtttagtaaa tcacacaaaa atccagcagaaattgtgaaa 180 atcctgaaag acaatttggc cattttggaa aagcaagaca aaaagacagacaaggcttca 240 gaagaagtgt ctaaatcact gcaagcaatg aaagaaattc tgtgtggtacaaacgagaaa 300 gaacccccga cagaagcagt ggctcagcta gcacaagaac tctacagcagtggcctgctg 360 gtgacactga tagctgacct gcagctgata gactttgagg gaaaaaaagatgtgacccag 420 atatttaaca acatcttgag aagacagata ggcactcgga gtcctactgtggagtatatt 480 agtgctcatc ctcatatcct gtttatgctc ctcaaaggat atgaagccccacagattgcc 540 ttacgttgtg ggattatgct gagagaatgt attcgacatg aaccacttgccaaaatcatc 600 ctcttttcta atcaattcag agatttcttt aagtacgtgg agttgtcaacatttgatatt 660 gcttcagatg cctttgctac tttcaaggat ttactaacca gacataaagtgttggtagca 720 gacttcttag aacaaaatta cgacactatt tttgaagact atgagaaattgcttcagtct 780 gagaattatg ttactaagag acagtcttta aagctgctag gggagctgatcctggaccgt 840 cacaactttg ccatcatgac aaagtatatc agcaagccgg agaacctgaaactcatgatg 900 aacctccttc gggataaaag tcccaacatc cagtttgaag cctttcatgtttttaaggtg 960 tttgtggcca gtcctcacaa aacacagcct attgtggaga tcctgttaaaaaatcagccc 1020 aaactcattg agtttctgag cagcttccaa aaagaaagga cggatgatgagcagttcgct 1080 gacgagaaga actacttgat taaacagatc cgagacttga agaaaacggccccttgaaga 1140 gctccccggc ccctgtcaca gtcagtcgtc tcatttgtcc agtttgtacagtgtgtcatt 1200 tcagaaagtc atcattcttg ggaagacttt ggaggtgcct attttttctgctgtaattgt 1260 tctgggtaga tggagtataa acatttgaat ggaaaaaaat taacctagaataatatattc 1320 attttagtca aaaaaaaaaa aaaa 1344 3 341 PRT Mus sp.g262934 3 Met Pro Phe Pro Phe Gly Lys Ser His Lys Ser Pro Ala Asp Ile 15 10 15 Val Lys Asn Leu Lys Glu Ser Met Ala Val Leu Glu Lys Gln Asp 2025 30 Ile Ser Asp Lys Lys Ala Glu Lys Ala Thr Glu Glu Val Ser Lys 35 4045 Asn Leu Val Ala Met Lys Glu Ile Leu Tyr Gly Thr Asn Glu Lys 50 55 60Glu Pro Gln Thr Glu Ala Val Ala Gln Leu Ala Gln Glu Leu Tyr 65 70 75 AsnSer Gly Leu Leu Gly Thr Leu Val Ala Asp Leu Gln Leu Ile 80 85 90 Asp PheGlu Gly Lys Lys Asp Val Ala Gln Ile Phe Asn Asn Ile 95 100 105 Leu ArgArg Gln Ile Gly Thr Arg Thr Pro Thr Val Glu Tyr Ile 110 115 120 Cys ThrGln Gln Asn Ile Leu Phe Met Leu Leu Lys Gly Tyr Glu 125 130 135 Ser ProGlu Ile Ala Leu Asn Cys Gly Ile Met Leu Arg Glu Cys 140 145 150 Ile ArgHis Glu Pro Leu Ala Lys Ile Ile Leu Trp Ser Glu Gln 155 160 165 Phe TyrAsp Phe Phe Arg Tyr Val Glu Met Ser Thr Phe Asp Ile 170 175 180 Ala SerAsp Ala Phe Ala Thr Phe Lys Asp Leu Leu Thr Arg His 185 190 195 Lys LeuLeu Ser Ala Glu Phe Leu Glu Gln His Tyr Asp Arg Phe 200 205 210 Phe SerGlu Tyr Glu Lys Leu Leu His Ser Glu Asn Tyr Val Thr 215 220 225 Lys ArgGln Ser Leu Lys Leu Leu Gly Glu Leu Leu Leu Asp Arg 230 235 240 His AsnPhe Thr Ile Met Thr Lys Tyr Ile Ser Lys Pro Glu Asn 245 250 255 Leu LysLeu Met Met Asn Leu Leu Arg Asp Lys Ser Arg Asn Ile 260 265 270 Gln PheGlu Ala Phe His Val Phe Lys Val Phe Val Ala Asn Pro 275 280 285 Asn LysThr Gln Pro Ile Leu Asp Ile Leu Leu Lys Asn Gln Thr 290 295 300 Lys LeuIle Glu Phe Leu Ser Lys Phe Gln Asn Asp Arg Thr Glu 305 310 315 Asp GluGln Phe Asn Asp Glu Lys Thr Tyr Leu Val Lys Gln Ile 320 325 330 Arg AsnLeu Lys Arg Ala Ala Gln Gln Glu Ala 335 340 4 339 PRT Drosophilamelanogaster g1794137 4 Met Pro Leu Phe Gly Lys Ser Gln Lys Ser Pro ValGlu Leu Val 1 5 10 15 Lys Ser Leu Lys Glu Ala Ile Asn Ala Leu Glu AlaGly Asp Arg 20 25 30 Lys Val Glu Lys Ala Gln Glu Asp Val Ser Lys Asn LeuVal Ser 35 40 45 Ile Lys Asn Met Leu His Gly Ser Ser Asp Ala Glu Pro ProAla 50 55 60 Asp Tyr Val Val Ala Gln Leu Ser Gln Glu Leu Tyr Asn Ser Asn65 70 75 Leu Leu Leu Leu Leu Ile Gln Asn Leu His Arg Ile Asp Phe Glu 8085 90 Gly Lys Lys His Val Ala Leu Ile Phe Asn Asn Leu Leu Arg Arg 95 100105 Gln Ile Gly Thr Arg Ser Pro Thr Val Glu Tyr Ile Cys Thr Lys 110 115120 Pro Glu Ile Leu Phe Thr Leu Met Ala Gly Tyr Glu Asp Ala His 125 130135 Pro Glu Ile Ala Leu Asn Ser Gly Thr Met Leu Arg Glu Cys Ala 140 145150 Arg Tyr Glu Ala Leu Ala Lys Ile Met Leu His Ser Asp Glu Phe 155 160165 Phe Lys Phe Phe Arg Tyr Val Glu Val Ser Thr Phe Asp Ile Ala 170 175180 Ser Asp Ala Phe Ser Thr Phe Lys Glu Leu Leu Thr Arg His Lys 185 190195 Leu Leu Cys Ala Glu Phe Leu Asp Ala Asn Tyr Asp Lys Phe Phe 200 205210 Ser Gln His Tyr Gln Arg Leu Leu Asn Ser Glu Asn Tyr Val Thr 215 220225 Arg Arg Gln Ser Leu Lys Leu Leu Gly Glu Leu Leu Leu Asp Arg 230 235240 His Asn Phe Thr Val Met Thr Arg Tyr Ile Ser Glu Pro Glu Asn 245 250255 Leu Lys Leu Met Met Asn Met Leu Lys Glu Lys Ser Arg Asn Ile 260 265270 Gln Phe Glu Ala Phe His Val Phe Lys Val Phe Val Ala Asn Pro 275 280285 Asn Lys Pro Lys Pro Ile Leu Asp Ile Leu Leu Arg Asn Gln Thr 290 295300 Lys Leu Val Asp Phe Leu Thr Asn Phe His Thr Asp Arg Ser Glu 305 310315 Asp Glu Gln Phe Asn Asp Glu Lys Ala Tyr Leu Ile Lys Gln Ile 320 325330 Lys Glu Leu Lys Pro Leu Pro Glu Ala 335 5 377 PRT Caenorhabditiselegans g1255838 5 Met Pro Leu Leu Phe Gly Lys Ser His Lys Ser Pro AlaAsp Val 1 5 10 15 Val Lys Thr Leu Arg Glu Val Leu Thr Ile Leu Asp LysLeu Pro 20 25 30 Pro Pro Lys Leu Asp Lys Asp Gly Asn Ile Gln Ser Asp LysLys 35 40 45 Tyr Asp Lys Ala Leu Asp Glu Val Ser Lys Asn Val Ala Met Ile50 55 60 Lys Ser Phe Ile Tyr Gly Asn Asp Ser Ala Glu Pro Ser Ser Glu 6570 75 His Val Val Gln Val Ala Gln Leu Ala Gln Glu Val Tyr Asn Ala 80 8590 Asn Ile Leu Pro Met Leu Ile Lys Met Leu Pro Lys Phe Glu Phe 95 100105 Glu Cys Lys Lys Asp Val Gly Gln Ile Phe Asn Asn Leu Leu Arg 110 115120 Arg Gln Ile Gly Thr Arg Ser Pro Thr Val Glu Tyr Leu Gly Ala 125 130135 Arg Pro Glu Ile Leu Ile Gln Leu Val Gln Gly Tyr Ser Val Pro 140 145150 Asp Ile Ala Leu Thr Cys Gly Leu Met Leu Arg Glu Ser Ile Arg 155 160165 His Asp His Leu Ala Lys Ile Ile Leu Tyr Ser Asp Val Phe Tyr 170 175180 Thr Phe Phe Leu Tyr Val Gln Ser Glu Val Phe Asp Ile Ser Ser 185 190195 Asp Ala Phe Ser Thr Phe Lys Glu Leu Thr Thr Arg His Lys Ala 200 205210 Ile Ile Ala Glu Phe Leu Asp Ser Asn Tyr Asp Thr Phe Phe Ala 215 220225 Gln Tyr Gln Asn Leu Leu Asn Ser Lys Asn Tyr Val Thr Arg Arg 230 235240 Gln Ser Leu Lys Leu Leu Gly Glu Leu Leu Leu Asp Arg His Asn 245 250255 Phe Asn Thr Met Thr Lys Tyr Ile Ser Asn Pro Asp Asn Leu Arg 260 265270 Leu Met Met Glu Leu Leu Arg Asp Lys Ser Arg Asn Ile Gln Tyr 275 280285 Glu Ala Phe His Val Phe Lys Val Phe Val Ala Asn Pro Asn Lys 290 295300 Pro Lys Pro Ile Ser Asp Ile Leu Asn Arg Asn Arg Glu Lys Leu 305 310315 Val Glu Phe Leu Ser Glu Phe His Asn Asp Arg Thr Asp Asp Glu 320 325330 Gln Phe Asn Asp Glu Lys Ala Tyr Leu Ile Lys Gln Ile Gln Glu 335 340345 Met Lys Ser Ser Pro Lys Glu Ala Lys Lys Pro Lys Ser Lys Glu 350 355360 Asp Glu Asn Gln Glu Pro Ala Gly Pro Ser Glu Gly Pro Ser Thr 365 370375 Ser Gln

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: a) a polypeptide comprising an amino acid sequence of SEQID NO:1, b) a polypeptide comprising a naturally occurring amino acidsequence at least 90% identical to an amino acid sequence of SEQ IDNO:1, c) a biologically active fragment of a polypeptide having an aminoacid sequence of SEQ ID NO:1, and d) an immunogenic fragment of apolypeptide having an amino acid sequence of SEQ ID NO:1.
 2. An isolatedpolypeptide of claim 1, having a sequence of SEQ ID NO:1.
 3. An isolatedpolynucleotide encoding a polypeptide of claim
 1. 4. An isolatedpolynucleotide encoding a polypeptide of claim
 2. 5. An isolatedpolynucleotide of claim 4, having a sequence of SEQ ID NO:2.
 6. Arecombinant polynucleotide comprising a promoter sequence operablylinked to a polynucleotide of claim
 3. 7. A cell transformed with arecombinant polynucleotide of claim
 6. 8. A transgenic organismcomprising a recombinant polynucleotide of claim
 6. 9. A method forproducing a polypeptide of claim 1, the method comprising: a) culturinga cell under conditions suitable for expression of the polypeptide,wherein said cell is transformed with a recombinant polynucleotide, andsaid recombinant polynucleotide comprises a promoter sequence operablylinked to a polynucleotide encoding the polypeptide of claim 1, and b)recovering the polypeptide so expressed.
 10. A method of claim 9,wherein the polypeptide has the sequence of SEQ ID NO:1.
 11. An isolatedantibody which specifically binds to a polypeptide of claim
 1. 12. Anisolated polynucleotide selected from the group consisting of: a) apolynucleotide comprising a polynucleotide sequence of SEQ ID NO:2, b) apolynucleotide comprising a naturally occurring polynucleotide sequenceat least 90% identical to a polynucleotide sequence of SEQ ID NO:2, c) apolynucleotide complementary to a polynucleotide of a), d) apolynucleotide complementary to a polynucleotide of b), and e) an RNAequivalent of a)-d).
 13. An isolated polynucleotide comprising at least60 contiguous nucleotides of a polynucleotide of claim
 12. 14. A methodfor detecting a target polynucleotide in a sample, said targetpolynucleotide having a sequence of a polynucleotide of claim 12, themethod comprising: a) hybridizing the sample with a probe comprising atleast 20 contiguous nucleotides comprising a sequence complementary tosaid target polynucleotide in the sample, and which probe specificallyhybridizes to said target polynucleotide, under conditions whereby ahybridization complex is formed between said probe and said targetpolynucleotide or fragments thereof, and b) detecting the presence orabsence of said hybridization complex, and, optionally, if present, theamount thereof.
 15. A method of claim 14, wherein the probe comprises atleast 60 contiguous nucleotides.
 16. A method for detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide of claim 12, the method comprising: a) amplifyingsaid target polynucleotide or fragment thereof using polymerase chainreaction amplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent, the amount thereof.
 17. A composition comprising a polypeptideof claim 1 and a pharmaceutically acceptable excipient.
 18. Acomposition of claim 17, wherein the polypeptide has an amino acidsequence of SEQ ID NO:1.
 19. A method for treating a disease orcondition associated with decreased expression of functional HCBP,comprising administering to a patient in need of such treatment thecomposition of claim
 17. 20. A method for screening a compound foreffectiveness as an agonist of a polypeptide of claim 1, the methodcomprising: a) exposing a sample comprising a polypeptide of claim 1 toa compound, and b) detecting agonist activity in the sample.
 21. Acomposition comprising an agonist compound identified by a method ofclaim 20 and a pharmaceutically acceptable excipient.
 22. A method fortreating a disease or condition associated with decreased expression offunctional HCBP, comprising administering to a patient in need of suchtreatment a composition of claim
 21. 23. A method for screening acompound for effectiveness as an antagonist of a polypeptide of claim 1,the method comprising: a) exposing a sample comprising a polypeptide ofclaim 1 to a compound, and b) detecting antagonist activity in thesample.
 24. A composition comprising an antagonist compound identifiedby a method of claim 23 and a pharmaceutically acceptable excipient. 25.A method for treating a disease or condition associated withoverexpression of functional HCBP, comprising administering to a patientin need of such treatment a composition of claim
 24. 26. A method ofscreening for a compound that specifically binds to the polypeptide ofclaim 1, the method comprising: a) combining the polypeptide of claim 1with at least one test compound under suitable conditions, and b)detecting binding of the polypeptide of claim 1 to the test compound,thereby identifying a compound that specifically binds to thepolypeptide of claim
 1. 27. A method of screening for a compound thatmodulates the activity of the polypeptide of claim 1, said methodcomprising: a) combining the polypeptide of claim 1 with at least onetest compound under conditions permissive for the activity of thepolypeptide of claim 1, b) assessing the activity of the polypeptide ofclaim 1 in the presence of the test compound, and c) comparing theactivity of the polypeptide of claim 1 in the presence of the testcompound with the activity of the polypeptide of claim 1 in the absenceof the test compound, wherein a change in the activity of thepolypeptide of claim 1 in the presence of the test compound isindicative of a compound that modulates the activity of the polypeptideof claim
 1. 28. A method for screening a compound for effectiveness inaltering expression of a target polynucleotide, wherein said targetpolynucleotide comprises a polynucleotide sequence of claim 5, themethod comprising: a) exposing a sample comprising the targetpolynucleotide to a compound, under conditions suitable for theexpression of the target polynucleotide, b) detecting altered expressionof the target polynucleotide, and c) comparing the expression of thetarget polynucleotide in the presence of varying amounts of the compoundand in the absence of the compound.
 29. A method for assessing toxicityof a test compound, the method comprising: a) treating a biologicalsample containing nucleic acids with the test compound, b) hybridizingthe nucleic acids of the treated biological sample with a probecomprising at least 20 contiguous nucleotides of a polynucleotide ofclaim 12 under conditions whereby a specific hybridization complex isformed between said probe and a target polynucleotide in the biologicalsample, said target polynucleotide comprising a polynucleotide sequenceof a polynucleotide of claim 12 or fragment thereof, c) quantifying theamount of hybridization complex, and d) comparing the amount ofhybridization complex in the treated biological sample with the amountof hybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.
 30. Adiagnostic test for a condition or disease associated with theexpression of HCBP in a biological sample, the method comprising: a)combining the biological sample with an antibody of claim 11, underconditions suitable for the antibody to bind the polypeptide and form anantibody:polypeptide complex, and b) detecting the complex, wherein thepresence of the complex correlates with the presence of the polypeptidein the biological sample.
 31. The antibody of claim 11, wherein theantibody is: a) a chimeric antibody, b) a single chain antibody, c) aFab fragment, d) a F(ab′)₂ fragment, or e) a humanized antibody.
 32. Acomposition comprising an antibody of claim 11 and an acceptableexcipient.
 33. A method of diagnosing a condition or disease associatedwith the expression of HCBP in a subject, comprising administering tosaid subject an effective amount of the composition of claim
 32. 34. Acomposition of claim 32, wherein the antibody is labeled.
 35. A methodof diagnosing a condition or disease associated with the expression ofHCBP in a subject, comprising administering to said subject an effectiveamount of the composition of claim
 34. 36. A method of preparing apolyclonal antibody with the specificity of the antibody of claim 11,the method comprising: a) immunizing an animal with a polypeptide havingan amino acid sequence of SEQ ID NO:1, or an immunogenic fragmentthereof, under conditions to elicit an antibody response, b) isolatingantibodies from said animal, and c) screening the isolated antibodieswith the polypeptide, thereby identifying a polyclonal antibody whichbinds specifically to a polypeptide having an amino acid sequence of SEQID NO:1.
 37. An antibody produced by a method of claim
 36. 38. Acomposition comprising the antibody of claim 37 and a suitable carrier.39. A method of making a monoclonal antibody with the specificity of theantibody of claim 11, the method comprising: a) immunizing an animalwith a polypeptide having an amino acid sequence of SEQ ID NO:1, or animmunogenic fragment thereof, under conditions to elicit an antibodyresponse, b) isolating antibody producing cells from the animal, c)fusing the antibody producing cells with immortalized cells to formmonoclonal antibody-producing hybridoma cells, d) culturing thehybridoma cells, and e) isolating from the culture monoclonal antibodywhich binds specifically to a polypeptide having an amino acid sequenceof SEQ ID NO:1.
 40. A monoclonal antibody produced by a method of claim39.
 41. A composition comprising the antibody of claim 40 and a suitablecarrier.
 42. The antibody of claim 11, wherein the antibody is producedby screening a Fab expression library.
 43. The antibody of claim 11,wherein the antibody is produced by screening a recombinantimmunoglobulin library.
 44. A method of detecting a polypeptide havingan amino acid sequence of SEQ ID NO:1 in a sample, the methodcomprising: a) incubating the antibody of claim 11 with a sample underconditions to allow specific binding of the antibody and thepolypeptide, and b) detecting specific binding, wherein specific bindingindicates the presence of a polypeptide having an amino acid sequence ofSEQ ID NO:1 in the sample.
 45. A method of purifying a polypeptidehaving an amino acid sequence of SEQ ID NO:1 from a sample, the methodcomprising: a) incubating the antibody of claim 11 with a sample underconditions to allow specific binding of the antibody and thepolypeptide, and b) separating the antibody from the sample andobtaining the purified polypeptide having an amino acid sequence of SEQID NO:1.
 46. A microarray wherein at least one element of the microarrayis a polynucleotide of claim
 13. 47. A method of generating anexpression profile of a sample which contains polynucleotides, themethod comprising: a) labeling the polynucleotides of the sample, b)contacting the elements of the microarray of claim 46 with the labeledpolynucleotides of the sample under conditions suitable for theformation of a hybridization complex, and c) quantifying the expressionof the polynucleotides in the sample.
 48. An array comprising differentnucleotide molecules affixed in distinct physical locations on a solidsubstrate, wherein at least one of said nucleotide molecules comprises afirst oligonucleotide or polynucleotide sequence specificallyhybridizable with at least 30 contiguous nucleotides of a targetpolynucleotide, and wherein said target polynucleotide is apolynucleotide of claim
 12. 49. An array of claim 48, wherein said firstoligonucleotide or polynucleotide sequence is completely complementaryto at least 30 contiguous nucleotides of said target polynucleotide. 50.An array of claim 48, wherein said first oligonucleotide orpolynucleotide sequence is completely complementary to at least 60contiguous nucleotides of said target polynucleotide.
 51. An array ofclaim 48, wherein said first oligonucleotide or polynucleotide sequenceis completely complementary to said target polynucleotide.
 52. An arrayof claim 48, which is a microarray.
 53. An array of claim 48, furthercomprising said target polynucleotide hybridized to a nucleotidemolecule comprising said first oligonucleotide or polynucleotidesequence.
 54. An array of claim 48, wherein a linker joins at least oneof said nucleotide molecules to said solid substrate.
 55. An array ofclaim 48, wherein each distinct physical location on the substratecontains multiple nucleotide molecules, and the multiple nucleotidemolecules at any single distinct physical location have the samesequence, and each distinct physical location on the substrate containsnucleotide molecules having a sequence which differs from the sequenceof nucleotide molecules at another distinct physical location on thesubstrate.
 56. A polypeptide of claim 1, comprising the amino acidsequence of SEQ ID NO:1.
 57. A polynucleotide of claim 12, comprisingthe polynucleotide sequence of SEQ ID NO:2.